Glycoprotein biomarkers for esophageal adenocarcinoma and barrett&#39;s esophagus and uses thereof

ABSTRACT

Disclosed are biomarkers for Barrett&#39;s esophagus and esophageal adenocarcinoma, and uses thereof, such as in methods for detecting the presence, and monitoring progression, of Barrett&#39;s esophagus and esophageal adenocarcinoma. Also disclosed are methods for treating and methods of monitoring the treatment of Barrett&#39;s esophagus and esophageal adenocarcinoma, as well as kits and compositions for use in such methods.

RELATED APPLICATIONS

This application is the U.S. National Stage Application under 35 U.S.C.§ 371 of International Application No. PCT/AU2015/050723, filed Nov. 17,2015, designating the U.S. and published as WO 2016/077881 A1 on May 26,2016, which claims priority to Australian Provisional Application No.2014904616 entitled “Biomarkers and Uses Therefor”, filed on Nov. 17,2014. Any and all applications for which a foreign or a domesticpriority is claimed is/are identified in the Application Data Sheetfiled herewith and is/are hereby incorporated by reference in theirentirety under 37 C.F.R. § 1.57.

FIELD OF THE INVENTION

This invention relates generally to biomarkers for Barrett's esophagusand esophageal adenocarcinoma, and uses thereof, such as in methods fordetecting the presence, and monitoring progression, of Barrett'sesophagus and esophageal adenocarcinoma. The invention also relates tomethods for treating and methods of monitoring the treatment ofBarrett's esophagus and esophageal adenocarcinoma, as well as to kitsand compositions for use in such methods.

BACKGROUND OF THE INVENTION

Esophageal adenocarcinoma (EAC) is now the leading esophageal cancer inindustrialized countries, with an alarming increase in incidence of 3%per year over the past 30 years. Although EAC is rare in subjects under40 years of age, its incidence increases significantly with each decadethereafter. This is likely a result of changing lifestyle and foodhabits, with known EAC risk factors including, for example, accumulationof abdominal visceral fat, high dietary fat and cholesterol intake withlow fruit and vegetable intake, acid reflux (gastroesophageal refluxdisease), and smoking.

Barrett's esophagus (BE; also known as Barrett's metaplasia), ametaplastic change to the esophageal lining characterized by replacementof normal stratified squamous epithelium with metaplastic columnarepithelium, is a major risk factor for EAC and most EAC is thought todevelop from BE. Subjects with BE have 30-125 times greater risk ofdeveloping EAC than non-BE subjects, and it is estimated that 0.5% to 1%of subjects with BE develop EAC each year (Tischoff et al. Expert RevGastroenterol Hepatol 2008; 2:653-63). BE develops relatively slowly,generally over 5 to 10 years. This development is thought to be inresponse to chronic gastroesophageal reflux disorder, which is a commoncondition in western populations. The malignant progression to EACfollows a generally accepted series of stages, from metaplasia, tolow-grade dysplasia (LGD), to high-grade dysplasia (HGD), and then toadenocarcinoma, with the involvement of genetic and epigeneticmodifications.

Despite aggressive treatment, the 5-year survival rate for EAC is low atjust 9 to 24%. This is likely due to late stage diagnosis: approximatelytwo thirds of patients who are diagnosed have advanced-stage disease, atwhich point current therapies are largely ineffective. Current screeningprotocols for EAC generally involve endoscopic screening of patientswith high risk chronic gastroesophageal reflux disorder to determine thedegree of dysplasia in endoscopic biopsy samples. Those with HGD arecandidates for endoscopic mucosal ablation or esophageal resection toslow or prevent disease progression. However, these endoscopic screeningprograms have limitations associated with sampling error, variability inassessment of biopsies between practitioners, and tissue heterogeneity.False positives occur and, conversely, invasive cancer has been found inup to 40% of patients despite negative endoscopic results. Moreover,even with these endoscopic screening programs, more than 80% of EACs arediagnosed without any prior diagnosis of BE or gastroesophageal refluxdisorder, more than 80% of patients with BE are undiagnosed (and thusnot recommended for subsequent and ongoing screening programs), and manypatients undergoing routine screening never progress to EAC. Thisindicates that current screening methodologies are not particularlyeffective at identifying patients at high risk and distinguishingbetween those that progress to EAC and those that don't. Hence, there isan unmet clinical need for improved methods for EAC diagnosis.

SUMMARY OF THE INVENTION

The present invention is predicated in part on the identification ofserum glycoproteins that are differentially glycosylated in subjectswith EAC, BE and in healthy subjects (i.e., subjects having a healthycondition (HC)). Thus, subjects with EAC have a different serumglycosylation “signature” or “profile” than patients with BE and healthypatients, and patients with BE have different a serum glycosylationsignature or profile than healthy patients. Accordingly, as describedherein, detecting the level of one or more of these different types ofglycosylation in a biological sample, such as a blood, serum or plasmasample, from a subject can be used to determine the likelihood ofpresence of absence of either EAC or BE in the subject. Monitoring thelevels of one or more of the types of glycosylation identified hereincan also be used to monitor the progress of EAC or BE, such as before,during and/or after treatment. Accordingly, in some aspects, monitoringthe levels of one or more types of glycosylation identified herein canalso be used to monitor the efficacy of treatment of EAC or BE.

The present invention thus represents a significant advance over currenttechnologies for the management of EAC and BE. In certain advantageousembodiments, it relies upon measuring the level of at least oneglycospecies. The present invention also provides robust biomarkers fordetermining the likelihood of the presence or absence of EAC or BE.

In one aspect, the invention provides a method for determining thelikelihood of the presence or absence of a condition selected from a HC,EAC and BE in a subject, the method comprising determining in a samplefrom the subject the level of a glycospecies of a glycoprotein, whichglycospecies is differentially expressed between at least two of HC, EACand BE, and determining a likelihood of the subject having or not havingthe condition based on whether the level of the glycospecies is above orbelow a predetermined threshold that correlates with the presence of thecondition.

In some embodiments, the methods determine the likelihood that EAC ispresent or absent in a subject, wherein the method comprises determiningin a sample from the subject the level of a glycospecies of aglycoprotein, which glycospecies is differentially expressed between EACand one or more other conditions (e.g., a HC and/or BE), and determiningthe likelihood of EAC being present or absent in the subject based onwhether the level of the glycospecies is above or below a predeterminedthreshold that correlates with the presence or absence of EAC.

In some embodiments, the methods determine the likelihood that BE ispresent or absent in a subject, wherein the methods comprise determiningin a sample from the subject the level of a glycospecies of aglycoprotein, which glycospecies is differentially expressed between BEand one or more other conditions (e.g., a HC and/or EAC), anddetermining a likelihood of BE being present or absent in the subjectbased on whether the level of the glycospecies is above or below apredetermined threshold that correlates with the presence or absence ofBE.

In some embodiments, the methods determine the likelihood of the subjecthaving a HC in a subject, that is the absence of EAC and/or BE. In suchembodiments, the methods comprise determining in a sample from thesubject the level of a glycospecies of a glycoprotein, whichglycospecies is differentially expressed between HC and one or moreother conditions (e.g., BE and/or EAC), and determining the likelihoodof the subject having or not having a HC based on whether the level ofthe glycospecies is above or below a predetermined threshold thatcorrelates with the presence or absence of a HC.

In another aspect, the invention provides methods for determining thelikelihood of the presence or absence of a condition selected from a HC,EAC and BE in a subject, the method comprising determining in a samplefrom the subject the respective levels of a plurality of glycospecies ofone or more glycoproteins, wherein individual glycospecies aredifferentially expressed between at least two of HC, EAC and BE, anddetermining a likelihood of the subject having or not having thecondition based on whether the respective levels of the individualglycospecies are above or below a corresponding predetermined thresholdthat correlates with the presence or absence of the condition.

In some embodiments of this aspect, the methods determine the likelihoodof EAC being present or absent in a subject, wherein the methodscomprise determining in a sample from the subject the respective levelsof a plurality of glycospecies of one or more glycoproteins, whereinindividual glycospecies are differentially expressed between EAC and oneor more other conditions (e.g., a HC and/or BE), and determining alikelihood of the subject having or not having EAC based on whether therespective levels of the individual glycospecies are above or below acorresponding predetermined threshold that correlates with the presenceor absence of EAC.

In some embodiments, the methods determine the likelihood of BE beingpresent or absent in a subject, in which the methods comprisedetermining in a sample from the subject the respective levels of aplurality of glycospecies of one or more glycoproteins, whereinindividual glycospecies are differentially expressed between BE and oneor more other conditions (e.g., a HC and/or EAC), and determining alikelihood of the subject having or not having BE based on whether therespective levels of the individual glycospecies are above or below acorresponding predetermined threshold that correlates with the presenceor absence of BE.

In other embodiments, the methods for determining the likelihood of thepresence or absence of a HC in a subject comprise determining in asample from the subject the respective levels of a plurality ofglycospecies of one or more glycoproteins, wherein individualglycospecies are differentially expressed between HC and one or moreother conditions (e.g., BE and/or EAC), and determining a likelihood ofthe subject having or not having HC based on whether the respectivelevels of the individual glycospecies are above or below a correspondingpredetermined threshold that correlates with the presence or absence ofHC.

In some embodiments, an individual glycoprotein comprises a firstglycospecies and a second glycospecies wherein the first glycospecies isdifferentially expressed between a plurality of conditions and thesecond glycospecies is not so differentially expressed.

In some preferred embodiments the glycoprotein is selected from thegroup comprising or consisting of: afamin, alpha-1-antichymotrypsin,alpha-1-antitrypsin, alpha-1-acid glycoprotein 1, alpha-1B-glycoprotein,alpha-2-antiplasmin, alpha-2-HS-glycoprotein, alpha-2-macroglobulin,alpha-2-antiplasmin, antithrombin-III, apolipoprotein B-100,beta-2-glycoprotein 1, C4b-binding protein alpha chain, ceruloplasmin,coagulation factor XII, complement C1q subcomponent subunit B,complement C5, complement component C7, complement component C9,complement factor B, ficolin-3, gelsolin, haptoglobin, hemopexin, plasmaprotease C1 inhibitor, serum paraoxonase/arylesterase 1, andserotransferrin.

In some embodiments, the level of an individual glycospecies isdetermined by contacting the sample with a glycan-binding moleculespecific for the glycospecies, under conditions that permit binding ofthe glycan-binding molecule to the glycospecies. The glycan-bindingmolecule is suitably selected from the group consisting of a lectin, aglycospecific antibody, a glycospecific aptamer, a glycospecificpeptide, and a glycospecific small molecule.

Illustrative lectins suitable for this purpose include Aleuria aurantialectin (AAL); erythroagglutinating phytohemagglutinin (EPHA); jacalin(JAC); Narcissus pseudonarcissus lectin (NPL); Pisum sativum agglutinin(PSA); wheat germ agglutinin (WGA); Bauhinia purpurea lectin (BPL);Erythrina cristagalli agglutinin (ECA); soybean agglutinin (SBA); Helixpomatia agglutinin (HPA); Wisteria floribunda agglutinin (WFA); Daturastramonium lectin (DSA); Helix aspersa agglutinin (HAA); Solanumtuberosum lectin (STL); concanavalin A (ConA); Galanthus nivalis lectin(GNL); Ulex europeus agglutinin-I (UEA); Maackia amurensis agglutinin-II(MAA), Sambucus nigra agglutinin (SNA); and leukoagglutinatingphytohemagglutinin (LPHA).

In some embodiments, glycospecies that are differentially expressedbetween subjects with EAC and healthy subjects are selected from thegroup comprising or consisting of: complement component AAL-bindingcomplement component C9, EPHA-binding complement component C9,JAC-binding complement component C9, NPL-binding complement componentC9, PSA-binding complement component C9, WGA-binding complementcomponent C9, AAL-binding gelsolin, EPHA-binding gelsolin, JAC-bindinggelsolin, PSA-binding gelsolin, EPHA-binding haptoglobin, NPL-bindinghaptoglobin, PSA-binding haptoglobin, WGA-binding haptoglobin,JAC-binding complement factor B, JAC-binding alpha- 1-antichymotrypsin,NPL-binding alpha-1 -antichymotrypsin, WGA-binding alpha-1-antichymotrypsin, JAC-binding complement C5, JAC-binding hemopexin,JAC-binding C4b-binding protein alpha chain, NPL-binding C4b-bindingprotein alpha chain, JAC-binding plasma protease C1 inhibitor,JAC-binding hemopexin, AAL-binding alpha-1-acid glycoprotein 1,EPHA-binding alpha-1-acid glycoprotein 1, JAC-binding ceruloplasmin,NPL-binding ceruloplasmin, NPL-binding antithrombin-III, STL-bindingficolin-3, WGA-binding complement C1q subcomponent subunit B.

Preferably, the glycospecies (i.e., defined by the glycan-bindingmolecule (e.g., lectin) and the glycoprotein to which it binds) that aredifferentially expressed between EAC and HC are selected from TABLE 1:

TABLE 1 Glycoprotein Lectin Overexpressed Underexpressed AAL complementcomponent C9 gelsolin PSA haptoglobin, complement component gelsolin C9EPHA haptoglobin, complement component gelsolin, alpha-2- C9macroglobulin, alpha- 2-HS-glycoprotein JAC complement factor B,alpha-1- gelsolin antichymotrypsin, complement C5, complement componentC9, hemopexin, C4b-binding protein alpha chain, plasma protease C1inhibitor, heparin cofactor 2 NPL haptoglobin, alpha-1-antichymotrypsin,complement component C9, C4b- binding protein alpha chain WGAhaptoglobin, alpha-1-antichymotrypsin, complement component C9

In specific embodiments, the glycospecies that are differentiallyexpressed between EAC and HC are selected from TABLE 2:

TABLE 2 Glycoprotein Lectin Overexpressed Underexpressed AAL complementcomponent C9 gelsolin EPHA haptoglobin, complement component gelsolin,alpha-2-HS- C9 glycoprotein JAC alpha-1-antichymotrypsin, gelsolincomplement component C9, plasma protease C1 inhibitor NPL haptoglobin,alpha-1- antichymotrypsin, complement component C9

In other embodiments, the glycospecies that are differentially expressedbetween EAC and HC are selected from the group comprising or consistingof those listed in TABLE 3:

TABLE 3 Glycoprotein Lectin Overexpressed Underexpressed AALalpha-1-antichymotrypsin, fibrinogen gamma chain, retinol-alpha-1-antitrypsin, binding protein 4, gelsolin, complement componentC9, vitamin K-dependent protein S, alpha-1B-glycoprotein, insulin-likegrowth factor- complement C1s binding protein complex acid subcomponent,complement labile subunit, N- component C7, alpha-1-acidacetylmuramoyl-L-alanine glycoprotein 2 amidase, afamin EPHAhaptoglobin, complement fibrinogen gamma chain, alpha- factor B,alpha-1- 2-HS-glycoprotein, gelsolin, antichymotrypsin, cadherin-5,haemoglobin subunit complement component C9, beta, N-acetylmuramoyl-L-complement C1s alanine amidase subcomponent, complement component C7 JAChaptoglobin, alpha-1- apolipoprotein A-I, fibrinogen antichymotrypsin,alpha-1- beta chain, retinol-binding antitrypsin, fibrinogen gammaprotein 4, plasma kallikrein, chain, complement component gelsolin,vitamin K-dependent C9, alpha-2-HS-glycoprotein, protein S, kallistatin,cadherin-5, plasma protease C1 inhibitor, insulin-like growth factor-complement C1s binding protein complex acid subcomponent labile subunit,centriolin, N- acetylmuramoyl-L-alanine amidase, haemoglobin subunitbeta, serotransferrin, fibrinogen gamma chain NPL haptoglobin, alpha-1-apolipoprotein A-I, alpha-1B- antichymotrypsin, alpha-1- glycoprotein,gelsolin, vitamin antitrypsin, complement K-dependent protein S, serumcomponent C9, paraoxonase/arylesterase 1, serotransferrin, hemopexin,cadherin-5, insulin-like growth alpha-1B-glycoprotein factor-bindingprotein complex acid labile subunit, alpha-1-acid glycoprotein 2,haemoglobin subunit beta, serum paraoxonase/lactonase 3, N-acetylmuramoyl-L-alanine amidase

In yet other embodiments, the glycospecies that are differentiallyexpressed between subjects with a HC and those with BE are selected fromthe group comprising or consisting of: EPHA-bindingalpha-2-macroglobulin, JAC-binding apolipoprotein B-100, NPL-bindingapolipoprotein B-100, AAL-binding ficolin-3, STL-binding ficolin-3,AAL-binding complement C1q subcomponent subunit C, EPHA-binding proteinAMBP, EPHA-binding alpha-1-acid glycoprotein 1, JAC-binding coagulationfactor XII, NPL-binding antithrombin-III, SNA-bindingalpha-2-antiplasmin, and STL-binding ceruloplasmin

Preferably, the glycospecies (i.e., defined by the glycan-bindingmolecule (e.g., lectin) and the glycoprotein to which it binds) that aredifferentially expressed between BE and a HC are selected from TABLE 4:

TABLE 4 Glycoprotein Lectin Overexpressed Underexpressed EPHAalpha-2-macroglobulin JAC apolipoprotein B-100 NPL apolipoprotein B-100

In specific embodiments, the glycospecies that are differentiallyexpressed between EAC and HC are selected from JAC-bindingapolipoprotein B-100 and NPL-binding apolipoprotein B-100.

In other embodiments, the glycospecies that are differentially expressedbetween subjects with BE and those with a HC are selected from thoselisted in TABLE 5:

TABLE 5 Glycoprotein Lectin Overexpressed Underexpressed AAL lumicanEPHA lumican Prothrombin, fibronectin JAC apolipoprotein M, prothrombin,angiotensinogen, apolipoprotein A- I, fibrinogen beta chain, plasmakallikrein, apolipoprotein B-100, histidine-rich glycoprotein, vitaminK-dependent protein S, complement component C8 alpha chain, complementfactor H, alpha- 2-antiplasmin, kallistatin, afamin, hemoglobin subunitbeta. NPL beta-2-glycoprotein 1 apolipoprotein B-100, alpha-2-antiplasmin, cadherin-5, hemoglobin subunit beta

In yet other embodiments, glycospecies that are differentially expressedbetween subjects with EAC and subjects with BE are selected from thegroup comprising or consisting of: AAL-binding complement component C9,EPHA-binding complement component C9, WGA-binding complement componentC9, JAC-binding complement component C9, NPL-binding complementcomponent C9, PSA-binding complement component C9, AAL-binding gelsolin,EPHA-binding gelsolin, JAC-binding gelsolin, PSA-binding gelsolin,NPL-binding gelsolin, WGA-binding gelsolin, AAL-binding haptoglobin,EPHA-binding haptoglobin, JAC-binding haptoglobin, PSA-bindinghaptoglobin, WGA-binding haptoglobin, JAC-binding complement factor B,EPHA-binding alpha-1-antichymotrypsin, PSA-bindingalpha-1-antichymotrypsin, JAC-binding alpha-l-antichymotrypsin,AAL-binding complement CS, JAC-binding complement CS, PSA-bindingcomplement CS, AAL-binding complement component C7, PSA-bindingcomplement component C7, EPHA-binding complement component C7,JAC-binding complement component C7, AAL-binding apolipoprotein B-100,NPL-binding apolipoprotein B-100, EPHA-binding serotransferrin,JAC-binding alpha-1-antitrypsin, JAC-binding alpha-1B-glycoprotein,AAL-binding alpha-1-acid glycoprotein 1, AAL-binding ficolin-3,AAL-binding complement C1q subcomponent subunit C, AAL-bindingalpha-1-acid glycoprotein 1, JAC-binding ceruloplasmin, STL-bindingceruloplasmin, JAC-binding coagulation factor XII, and SNA-bindingalpha-2-antiplasmin.

Preferably, the glycospecies (i.e., defined by the glycan-bindingmolecule (e.g., lectin) and the glycoprotein to which it binds) that aredifferentially expressed between EAC and BE are selected from TABLE 6:

TABLE 6 Glycoprotein Lectin Overexpressed Underexpressed AALhaptoglobin, complement 5, apolipoprotein B-100, gelsolin complementcomponent C9, complement component C7 PSA haptoglobin, gelsolinalpha-1-antichymotrypsin, complement 5, complement component C9,complement component C7 EPHA haptoglobin, gelsolinalpha-1-antichymotrypsin, complement component C9, serotransferrin,complement component C7 JAC haptoglobin, gelsolin complement factor B,alpha-1-antitrypsin, alpha-1-antichymotrypsin, complement C5, complementcomponent C9, alpha-1B-glycoprotein, complement component C7 NPLcomplement component C9 apolipoprotein B-100, gelsolin, afamin WGAhaptoglobin, complement gelsolin component C9

In specific embodiments, the glycospecies that are differentiallyexpressed between EAC and BE are selected from AAL-binding complementcomponent C9, EPHA-binding complement component C9, JAC-bindingcomplement component C9, NPL-binding complement component C9,JAC-binding alpha- 1-antichymotrypsin, JAC-bindingalpha-1B-glycoprotein; NPL-binding gelsolin; and EPHA-binding gelsolin.

In other embodiments, the glycospecies that are differentially expressedbetween EAC and BE are selected from those listed in TABLE 7:

TABLE 7 Glycoprotein Lectin Overexpressed Underexpressed AALProthrombin, complement component Retinol-binding protein 4 C9 EPHAComplement factor B, complement beta-2-glycoprotein 1, component C9,gelsolin, lumican, serum paraoxonase/lactonase 3 JAC Ceruloplasmin,prothrombin, alpha-1- antichymotrypsin, complement component C9,hemopexin, alpha- 1B-glycoprotein, plasma protease C1 inhibitor,complement factor I, complement factor H, complement C1s subcomponent,C4b-binding protein beta chain, Inter-alpha-trypsin inhibitor heavychain H4 NPL Complement component C9 gelsolin

In some preferred embodiments, the methods of the invention comprisedetermining the level of 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 or more than 20 glycospecies of one or moreglycoproteins. For example, the one or more glycoproteins may optionallybe selected from the group comprising: complement component 9, gelsolin,alpha-1B-glycoprotein, angiotensinogen, and alpha-2-macroglobulin. Morespecifically, the glycospecies may suitably comprise complementcomponent 9 selected from the group comprising or consisting of:JAC-binding complement component 9, NPL-binding complement component 9and WGA-binding complement component 9. Alternatively, or in addition,the glycospecies may also comprise gelsolin selected from the groupcomprising or consisting of EPHA-binding gelsolin and SNA-bindinggelsolin. Alternatively, or in further addition to these glycospecies,the glycospecies may also be one or more of EPHA-bindingalpha-1B-glycoprotein, WGA-binding angiotensinogen, and NPL-bindingalpha-2-macroglobulin.

In some embodiments, the predetermined threshold of the method describedherein, represents the level of a corresponding glycospecies in a samplefrom a control subject, or represents a value above or below the levelof the glycospecies in a sample from a control subject, which levelcorrelates with the presence of the selected condition. For example, thecontrol subject may have EAC or BE. Alternatively, the control subjectis a healthy subject that does not have EAC or BE.

In yet another aspect, the invention provides a method for determiningthe likelihood of the presence or absence of a condition selected from aHC, EAC and BE in a subject, the method comprising determining in asample from the subject the ratio of a level of a glycospecies of aglycoprotein to the total level of the glycoprotein in the sample, whichglycospecies is differentially expressed between at least two of HC, EACand BE, and determining a likelihood of the subject having or not havingthe condition based on whether the ratio of the glycospecies is above orbelow a predetermined threshold that correlates with the presence orabsence of the condition.

In a related aspect, the methods determine the likelihood of EAC beingpresent or absent in a subject, in which the method comprisesdetermining in a sample from the subject the ratio of a level of aglycospecies of a glycoprotein to the total level of the glycoprotein inthe sample, which glycospecies is differentially expressed between EACand one or more other conditions (e.g., a HC and/or BE), and determininga likelihood of EAC being present or absent in the subject based onwhether the ratio is above or below a predetermined threshold thatcorrelates with the presence or absence of EAC.

In another related aspect, the methods determine the likelihood of BEbeing present or absent in a subject, wherein the methods comprisedetermining in a sample from the subject the ratio of a level of aglycospecies of a glycoprotein to the total level of the glycoprotein inthe sample, which glycospecies is differentially expressed between BEand one or more other condition (e.g., a HC and/or EAC), and determininga likelihood of the subject having or not having BE based on whether theratio is above or below a predetermined threshold that correlates withthe presence or absence of BE

In yet another related aspect, the methods determining the likelihood ofthe subject having or not having a HC, the method comprising determiningin a sample from the subject the ratio of a level of a glycospecies of aglycoprotein to the total level of the glycoprotein in the sample, whichglycospecies is differentially expressed between a HC and one or moreother conditions (e.g., BE and/or EAC), and determining a likelihood ofthe subject having or not having a HC based on whether the ratio isabove or below a predetermined threshold that correlates with thepresence or absence of a HC.

In preferred embodiments, the determination is made based on the levelsor ratios of a plurality of glycospecies of one or more glycoproteins.

In some embodiments, upon determining that the likelihood of EAC beingpresent in the subject is above the predetermined threshold, the subjectis suitably exposed to a treatment regimen for treating EAC. Thetreatment regimens optionally comprise surgery, radiotherapy orchemotherapy. For example, surgery to remove all or part of theesophagus may be performed.

In some embodiments, the determination method is performed by a personwho also exposes the subject to the treatment regimen. Alternatively,the sample from the subject can be provided to another person (e.g., aperson in a laboratory) to perform the determination method beforeproviding the results of the determination method to the person whoexposes the subject to the treatment regimen.

In another aspect, the invention provides a method of monitoring theprogression of EAC in a subject, comprising determining in a firstsample from the subject the level of a glycospecies of a glycoprotein,determining in a second sample from the subject the level of theglycospecies, wherein the second sample is taken at a later time thanthe first sample, and comparing the levels in the first and secondsample, wherein an increase or decrease in the level of the glycospeciesin the second sample compared to the first sample correlates with theprogression or regression of EAC.

In a related aspect, the invention also provides a method for monitoringthe progression of EAC in a subject, comprising: determining in a firstsample from the subject the respective levels of a plurality ofglycospecies of one or more glycoproteins, determining in a secondsample from the subject the respective levels of the plurality ofglycospecies, wherein the second sample is taken at a later time thanthe first sample, and comparing the respective levels in the first andsecond sample, wherein an increase or decrease in the level of theglycospecies in the second sample compared to the first samplecorrelates with the progression or regression of EAC.

In yet another related aspect, the invention provides a method formonitoring the progression of EAC in a subject, comprising determiningin a first sample from the subject the respective ratios of the levelsof a plurality of glycospecies of one or more glycoproteins to the totallevels of the one or more glycoproteins, determining in a second samplefrom the subject the respective ratios of the levels of the plurality ofglycospecies to the total levels of the glycoproteins, wherein thesecond sample is taken from the subject at a later time than the firstsample, and comparing the respective ratios in the first and secondsample, wherein an increase or decrease in the ratio of the glycospeciesin the second sample compared to the first sample correlates with theprogression or regression of EAC.

The present findings also enable methods of monitoring the efficacy of atreatment regimen for treating a condition selected from a HC, EAC andBE, and determining a subject's response to such treatment (e.g.,whether it is a positive or negative response to such treatment). Thus,in another aspect, a method is provided for monitoring the efficacy of aparticular treatment regimen in a subject towards a desired health state(e.g., HC), the method comprising: (1) providing a correlation of areference glycospecies profile with the likelihood of having HC, whereinthe reference glycospecies profile evaluates at least one glycospecies(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more glycospecies) of one ormore glycoproteins, (2) obtaining a corresponding glycospecies profileof a subject having HC, EAC or BE after treatment with a treatmentregimen, wherein a similarity of the subject's glycospecies profileafter treatment to the reference glycospecies profile indicates thelikelihood that the treatment regimen is effective for changing thehealth status of the subject to the desired health state.

Still another aspect of the present invention provides a method forcorrelating a reference glycospecies profile with an effective treatmentregimen for a condition selected from HC, EAC and BE, wherein thereference glycospecies profile evaluates at least one glycospecies(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more glycospecies), the methodcomprising: (a) determining a sample glycospecies profile from a subjectwith the condition prior to treatment, wherein the sample glycospeciesprofile evaluates for at least one glycospecies in the referenceglycospecies profile a corresponding glycospecies, and correlating thesample glycospecies profile with a treatment regimen that is effectivefor treating the condition in the subject.

In another aspect, the present invention provides a method fordetermining whether a treatment regimen is effective for treating asubject with a condition selected from HC, EAC and BE, the methodcomprising: (a) correlating a reference glycospecies profile prior totreatment with an effective treatment regimen for the condition, whereinthe reference glycospecies profile evaluates at least one glycospecies(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more glycospecies), and (b)obtaining a sample glycospecies profile from the subject aftertreatment, wherein the sample glycospecies profile evaluates for anindividual glycospecies in the reference glycospecies profile acorresponding glycospecies, and wherein the sample glycospecies profileafter treatment indicates whether the treatment regimen is effective fortreating the condition in the subject.

In a further aspect, the present invention provides a method forcorrelating a glycospecies profile with a positive or negative responseto a treatment regimen for a condition selected from HC, EAC and BE, themethod comprising: (a) obtaining a glycospecies profile from a subjectwith the condition following commencement of the treatment regimen,wherein the glycospecies profile evaluates at least one glycospecies(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more glycospecies), and (b)correlating the glycospecies profile from the subject with a positive ornegative response to the treatment regimen.

Another aspect of the present invention provides a method fordetermining a positive or negative response to a treatment regimen by asubject with a condition selected from HC, EAC and BE, the methodcomprising: (a) correlating a reference glycospecies profile with apositive or negative response to the treatment regimen, wherein thereference glycospecies profile evaluates at least one glycospecies(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more glycospecies), and (b)determining a sample glycospecies profile from the subject, wherein thesubject's sample glycospecies profile evaluates for an individualglycospecies in the reference glycospecies profile a correspondingglycospecies and indicates whether the subject is responding to thetreatment regimen.

In some embodiments, the methods for determining a positive or negativeresponse to a treatment regimen further comprise: determining a firstsample glycospecies profile from the subject prior to commencing thetreatment regimen, wherein the first sample glycospecies profileevaluates at least one glycospecies (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 ormore glycospecies), and comparing the first sample glycospecies profilewith a second sample glycospecies profile from the subject aftercommencement of the treatment regimen, wherein the second sampleglycospecies profile evaluates for an individual glycospecies in thefirst sample glycospecies profile a corresponding glycospecies.

Evaluation of glycospecies suitably includes determining the levels ofindividual glycospecies, which correlate with the presence of acondition, as broadly described above and elsewhere herein.

In yet another aspect, the present invention provides a method ofmonitoring or screening individuals characterized as being at anincreased risk of developing BE and/or EAC for the likelihood of thesubject having or not having EAC or BE. Illustrative known risk factorsfor developing either or these conditions are known to be males over 40years of age who are diagnosed as obese (i.e., identified asaccumulating high levels of abdominal visceral fat, dietary fat), havinga high cholesterol intake, suffering from acid reflux (gastroesophagealreflux disease), and smoking. Accordingly, the present invention furtherprovides a method of monitoring or screening a high risk subject for BEor EAC, the method comprising determining in a sample from the subjectthe level of a glycospecies of a glycoprotein, which glycospecies isdifferentially expressed between at least two of HC, EAC and BE, anddetermining a likelihood of the subject having or not having thecondition based on whether the level of the glycospecies is above orbelow a predetermined threshold that correlates with the presence orabsence of the condition. In some embodiments, a sample from the subjectcan be compared with a previous sample taken at an earlier date from thesubject to determine the change in the levels of the glycospecies overtime, and thus determine whether the likelihood of the subjectdeveloping BE or EAC is increasing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the (A) lectin magnetic bead array (LeMBA), (B)biomarker discovery, and (C)-(D) biomarker verification processes. Twoapproaches are shown in (B) for biomarker discovery. The group bindingdifference method identifies candidates that are present in one groupand absent in another group. Sparse partial least squares-discriminantanalysis (sPLS-DA) combined with stability analyses generate a rankedlist of candidates. The analysis illustrated in (D) shows LeMBA coupledwith western immunoblotting for protein level verification. (MS: massspectrometry; IB: immunoblotting; HC: healthy condition; DC: diseasecondition).

FIG. 2 demonstrates the outlier detection feature of GlycoSelector whichallows the visualization of experimental errors or bias present in thedata using four different graphical visualization tools. (A and E)Principal component analysis, (B and F) hierarchical clustering, (C andG) boxplot graphical outputs (wherein “total intensity” is normalizedand scaled) and (D and H) barplots of the coefficient of variation forBE/EAC biomarker discovery screen. Unique numbers on the graph indicatethe individual patient sample run. Run number 63 (arrow) in panel A to Dwas considered as an outlier based on the visualization tools. Thesample was re-analyzed on the mass spectrometer and outlier detectionwas performed again (panel E to H).

FIG. 3 shows biomarker discovery and orthogonal verification. Serumsamples from 29 patients (healthy-9, BE-10 and EAC-10) were screenedusing LeMBA-GlycoSelector pipeline. (A) sPLS-DA clustering: The sPLS-DAsample representation is based on the top 100 lectin-protein candidatesthat differentiates EAC (left group on graph) from BE (right group ongraph). (B) Stability analysis: Amongst the top 100 sPLS-DA candidates,82 candidates passed the stability criteria cut-off of 0.6 using 10-foldcross-validation analysis repeated 1000 times. (C) Overlap betweenlectin-protein candidates that differentiates BE from healthy, EAC fromBE and EAC from healthy phenotype. (D) Number of unique candidateproteins identified for each lectin in LeMBA-GlycoSelector analysis.Each of 20 lectins used for screening identified at least one uniqueprotein candidate (Clear bars—candidates differently expressed betweenHC and BE; grey bars: candidates differently expressed between BE andEAC; black bars: candidates differently expressed between HC and EAC).(E and F) Label-free proteomics relative quantitation results for AAL-HPand AAL-GSN, respectively. (E) AAL-HP (AAL-P00738) and (F) AAL-GSN(AAL-P06396) were the top two candidates identified using sPLS-DA andgroup binding difference tool respectively. (G and H) Normalizedintensity for AAL-HP and AAL-GSN using immunoblotting (H: healthy; B:BE; E: EAC; and C: control).

FIG. 4 shows LeMBA-MS data for Gelsolin as an example. Y-axis showsrelative abundance (note the log scale). X-axis shows the binding toeach of the 20 lectins, grouped into general reactivity groups. Boxedlectins show statistically significantly different binding between BEand EAC groups (*p<0.05, Student's t-test). (Clear bars: HC; grey bars:BE; black bars: EAC).

FIG. 5 shows a reduction of fucosylated gelsolin in EAC. (A) Immunoblotshowing similar total serum gelsolin levels. (B) Individual data for thetwo fucose-reactive lectins with reduced gelsolin binding in EAC.(Circle plots: HC; square plots: BE; triangle plots: EAC).

FIG. 6 provides proof-of concept data of exemplary glycospecies markerpanels. (A) ROC curve of multimarker panel for BE vs EAC; (B) ROC curveof multimarker panel for HC vs EAC; and (C) ROC curve of multimarkerpanel for HC vs BE.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “a glycospecies” means one glycospecies ormore than one glycospecies.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps. By“consisting of” is meant including, and limited to, whatever follows thephrase “consisting of”. Thus, the phrase “consisting of” indicates thatthe listed elements are required or mandatory, and that no otherelements may be present. By “consisting essentially of” is meantincluding any elements listed after the phrase, and limited to otherelements that do not interfere with or contribute to the activity oraction specified in the disclosure for the listed elements.

The term “control subject”, as used in the context of the presentinvention, may refer to a subject known to be affected with a diseasecondition (e.g., EAC or BE) (positive control), or to a subject known tobe not affected or diagnosed with the disease condition (negativecontrol), i.e., healthy. It should be noted that a control subject thatis known to be healthy, i.e., not suffering from the disease condition,may possibly suffer from another disease not tested/known. It is alsounderstood that control subjects and healthy controls include dataobtained and used as a standard, i.e. it can be used over and over againfor multiple different subjects. In other words, for example, whencomparing a subject sample to a control sample, the data from thecontrol sample could have been obtained in a different set ofexperiments, for example, it could be an average obtained from a numberof healthy subjects and not actually obtained at the time the data forthe subject was obtained.

The term “correlating” generally refers to determining a relationshipbetween one type of data with another or with a state. In variousembodiments, correlating a glycospecies profile with the presence orabsence of a condition (e.g., a condition selected from a HC, EAC andBE) comprises determining the presence, absence or amount of at leastone glycospecies in a subject that suffers from that condition, or inpersons known to be free of that condition. In specific embodiments, aprofile of glycospecies levels, absences or presences is correlated to aglobal probability or a particular outcome, using receiver operatingcharacteristic (ROC) curves.

By “corresponding glycoprotein” is meant a glycoprotein biomarker thatis structurally and/or functionally similar to or the same as aglycospecies biomarker. Representative corresponding glycoproteinsinclude other glycospecies of the glycoprotein, and isoforms of theglycoprotein without any glycosylation.

The term “differential expression” of glycospecies as used herein, meansqualitative and/or quantitative differences in the temporal and/or localglycospecies expression patterns, e.g., between a biological sampletaken from subjects with a condition as compared to a comparable sampletaken from subjects lacking the condition. Thus, a differentiallyexpressed glycospecies may qualitatively have its expression altered,including an activation or inactivation in, for example, a biologicalsample from a subject with a disease condition (e.g., EAC) or diseasesusceptible condition (e.g., BE) versus a biological condition from ahealthy subject. The difference in glycospecies expression may also bequantitative, e.g., in that expression is modulated, i.e., eitheroverexpressed, resulting in an increased amount of glycospecies, orunderexpressed, resulting in a decreased amount of glycospecies. Thedegree to which glycospecies expression differs need only be largeenough to be quantified via standard quantification or characterizationtechniques. For example, a glycospecies is differentially expressedbetween the samples if the amount of the glycospecies in one sample issignificantly different (i.e., p<0.05) from the amount of theglycospecies in the other sample. It should be noted that if theglycospecies is detectable in one sample and not detectable in theother, then the glycospecies can be considered to be differentiallypresent.

As used herein, the term “likelihood” is used as a measure of whethersubjects with a particular glycospecies profile actually have acondition (or not) based on a given mathematical model. An increasedlikelihood for example may be relative or absolute and may be expressedqualitatively or quantitatively. For instance, an increased risk may beexpressed as simply determining the subject's level of a givenglycospecies and placing the test subject in an “increased risk”category, based upon previous population studies. Alternatively, anumerical expression of the test subject's increased risk may bedetermined based upon glycospecies level analysis.

Most naturally occurring secreted proteins (or peptides) comprisecarbohydrate or saccharide moieties attached to the peptide via specificlinkages to a select number of amino acids along the length of theprimary peptide chain. Thus, many naturally occurring peptides aretermed “glycopeptides” or “glycoproteins” or are referred to as“glycosylated” proteins or peptides.

The predominant sugars found on glycoproteins are glucose, galactose,mannose, fucose, N-acetylgalactosamine (“GalNAc”), N-acetylglucosamine(“GlcNAc”) and sialic acid (e.g., N-acetylneuraminic acid (“NANA” or“NeuAc”, where “Neu” is neuraminic acid) and “Ac” refers to “acetyl”).The processing of the sugar groups occurs co-translationally in thelumen of the ER and continues in the Golgi apparatus for N-linkedglycoproteins.

The oligosaccharide structure attached to the protein backbone is knownas a “glycan” molecule. The glycan structures found in naturallyoccurring glycopeptides are typically divided into two classes,“N-linked glycans” or N-linked oligosaccharides” and “O-linked glycans”or O-linked oligosaccharides

Peptides expressed in eukaryotic cells typically comprise N-glycans.“N-glycans” are N-glycosylated at an amide nitrogen of an asparagine oran arginine residue in a protein via an N-acetylglucosamine residue.These “N-linked glycosylation sites” occur in the peptide primarystructure containing, for example, the amino acid sequenceasparagine-X-serine/threonine, where X is any amino acid residue exceptproline and aspartic acid.

A “glycan-binding molecule” refers to any molecule that is capable ofbinding to a glycan component of a glycoprotein. Typically, theglycan-binding molecule is glycospecies-specific (or glycospecific) inthat it selectively binds the glycan of one glycospecies of aglycoprotein but not another, such that it can be used to distinguishdifferent glycospecies of the glycoprotein. Glycan-binding molecules canbe natural or synthetic, and include, for example, lectins,glycospecific antibodies, glycospecific aptamers (e.g. RNA aptamer, DNAaptamer, or peptide aptamer), glycospecific peptides, and glycospecificsmall molecule.

As used herein, a “glycoprotein” refers to a protein having glycanstructures associated with the polypeptide backbone. Glycoproteins canbe associated with one or more types of glycosylation at a single ordifferent sites. Glycoproteins that differ with respect to type ofglycosylation generally have the same amino acid sequence or essentiallythe same amino acid sequence (e.g. isoforms, allelic variants and othervariants are considered to have essentially the same amino acidsequence), while the glycan structures associated with a particular typeof glycosylation differ by at least one glycan.

The term “glycospecies” refers to a glycoprotein with a distinct type ofglycosylation. The “type” or glycosylation is characterized by theglycans present in the glycosylations on the surface of theglycoprotein. For example, a glycosylation may comprise fucose-relatedglycans, mannose-related glycans, sialic acid glycans, etc. Aglycoprotein may be characterized as belonging to one, two, three, four,five or more than 5 different glycospecies (for example, a glycosylationmay comprise both a fucose-related glycan, a mannose-related glycan andsialic acid-based glycan).

As used herein, a “healthy” subject is a subject that does not have EACor BE.

As used herein, “level” with reference to a glycoprotein or glycospeciesrefers to the amount or concentration of the glycoprotein orglycospecies in a sample. The amount or concentration may be absolute ormay be relative, and can be determined using any method known in theart.

In this regard, the term “underexpressed” and the like refer to adownward deviation in the level of expression of an EAC biomarker ascompared to a baseline expression level of a corresponding EAC biomarkerin a control sample. The term “overexpressed” refers to an upwarddeviation in the level of glycospecies as compared to a baselineexpression level of a corresponding glycospecies in a control sample.

As used herein, the term “predetermined threshold” refers to a value,above or below which, indicates the presence of disease, such as EAC orBE or a healthy condition. For example, for the purposes of the presentinvention, a predetermined threshold may represent the level of aparticular glycospecies of a glycoprotein, or the ratio of the level ofa particular glycospecies of a glycoprotein to the total glycoproteinlevel, in a sample from an appropriate control subject, such as ahealthy subject or a subject with BE, or in pooled samples from multiplecontrol subjects or medians or averages of multiple control subjects.Thus, a level or ratio above or below the threshold indicates thepresence of EAC or BE, as taught herein. In other examples, apredetermined threshold may represent a value larger or smaller than thelevel or ratio determined for a control subject so as to incorporate afurther degree of confidence that a level or ratio above or below thepredetermined threshold is indicative of the presence of disease, suchas EAC or BE. For example, the predetermined threshold may represent theaverage or median level of a glycospecies in a group of controlsubjects, plus or minus 1, 2, 3 or more standard deviations. Thoseskilled in the art can readily determine an appropriate predeterminedthreshold based on analysis of biological samples from appropriatecontrol subjects.

The term “receiver operating characteristic (ROC) curves” means agraphical measure of sensitivity (y-axis) vs. 1—specificity (x-axis) fora clinical test. An important measure of the accuracy of the clinicaltest is the area under the ROC curve value (AUC value). If this area isequal to 1.0 then this test is 100% accurate because both thesensitivity and specificity are 1.0 so there are no false positives andno false negatives. On the other hand a test that cannot discriminatethat is the diagonal line from 0,0 to 1,1. The ROC area for this line is0.5. ROC curve areas (AUC-values) are typically between 0.5 and 1.0, butalso ROC values below 0.5 can—according to information theory—be asgood, if the result is interpreted inversely. Therefore, according tothe present invention an AUC-value close to 1 (e.g., 0.95) representsthe same good measure for a clinical test as an AUC-value close to 0(e.g., 0.05).

The terms “sample”, “biological sample”, and the like mean a materialknown or suspected of containing one or more glycospecies or otherEAC/BE biomarkers. A test sample can be used directly as obtained fromthe source or following a pretreatment to modify the character of thesample. The sample is suitably derived from blood or plasma fractions,including cell fractions (e.g., comprising tumor cells) or lysatesthereof, cell-free or cell-depleted fractions, and the like. The samplecan be treated prior to use, such as diluting viscous fluids, and thelike. Methods of treatment can involve filtration, distillation,extraction, concentration, inactivation of interfering components (e.g.,inhibiting nucleases such as RNases and DNases), the addition ofreagents, and the like

The terms “subject”, “individual” or “patient”, used interchangeablyherein, refer to any animal subject, particularly a mammalian subject,more particularly a human subject. In some embodiments, the subjectpresents with clinical signs of a condition as defined herein. As usedherein, the term “clinical sign”, or simply “sign”, refers to objectiveevidence of a disease present in a subject. Symptoms and/or signsassociated with diseases referred to herein and the evaluation of suchsigns are routine and known in the art. Examples of signs of diseasevary depending upon the disease. Signs of EAC may include tumorigenesis,metastasis, angiogenesis. Typically, whether a subject has a disease,and whether a subject is responding to treatment, may be determined byevaluation of signs associated with the disease.

The terms “treat” and “treating” as used herein, unless otherwiseindicated, refer to both therapeutic treatment and prophylactic orpreventative measures, wherein the object is to prevent, eitherpartially or completely, ameliorate or slow down (lessen) the targetedcondition or disorder (e.g., EAC or BE), or one or more symptomassociated therewith. The terms are also used herein to denote delayingthe onset of, inhibiting (e.g., reducing or arresting the growth of),alleviating the effects of, or prolonging the life of a patientsuffering from, cancer, in particular, EAC . Those in need of treatmentinclude those diagnosed with the disorder, those suspected of having thedisorder, those predisposed to have the disorder as well as those inwhom the disorder is to be prevented. Hence, the subject to be treatedherein may have been diagnosed as having the disorder or may bepredisposed or susceptible to the disorder. In some embodiments,treatment refers to the eradication, removal, modification, or controlof primary, regional, or metastatic cancer tissue that results from theadministration of one or more therapeutic agents according to themethods of the invention. In other embodiments, such terms refer to theminimizing or delaying the spread of cancer resulting from theadministration of one or more therapeutic agents to a subject with sucha disease. In other embodiments, such terms refer to elimination ofdisease causing cells. The term “treatment” as used herein, unlessotherwise indicated, refers to the act of treating.

As used herein, the term “treatment regimen” refers to prophylacticand/or prophylactic regimen (i.e., before the onset of EAC, e.g., if thesubject is affected with BE), or to a therapeutic regimen (i.e., afterthe onset of EAC). The term “treatment regimen” encompasses naturalsubstances and pharmaceutical agents (i.e., “drugs”) as well as anyother treatment regimen including but not limited to chemotherapy,radiotherapy, proton therapy, immunotherapy, hormone therapy,phototherapy, cryotherapy, cryosurgery, toxin therapy or pro-apoptosistherapy, high intensity focused ultrasound, dietary treatments, physicaltherapy or exercise regimens, surgical interventions, and combinationsthereof.

Those skilled in the art will appreciate that the aspects andembodiments described herein are susceptible to variations andmodifications other than those specifically described. It is to beunderstood that the disclosure includes all such variations andmodifications. The disclosure also includes all of the steps, features,compositions and compounds referred to or indicated in thisspecification, individually or collectively, and any and allcombinations of any two or more of said steps or features.

2. Biomarkers for BE and EAC and uses therefor

Glycosylation is a dynamic, post-translational modification that can bealtered during the development and progression of a cancer, such as EAC,or a precancerous condition, such as BE. As a result, the sameglycoprotein may be expressed both before and after oncogenictransformation, but the glycosylation of the glycoprotein before andafter oncogenic transformation may be different.

Differences in the type of glycosylation include the removal of a glycancomponent, the addition of a glycan component, a change in the glycancomponent such as the substitution of one glycan component for another,the change in the branching of glycans, and the rearrangement of one ormore glycan components on the glycoprotein, as where a glycan componentis shifted from one position on the polypeptide sequence to another.Differential glycosylation can be detected using any of method known inthe art, including but not limited to, methods that detect binding of aparticular type of glycosylation to a glycan-binding molecule, such as alectin, glycospecific antibody or glycospecific aptamer, as furtherdescribed herein, that is selective and/or specific for the particulartype of glycosylation. Differences in glycosylation can also be detectedspectroscopically. For example mass spectrometry can be used tocharacterize the glycan component and distinguish between differenttypes of glycosylation.

The present invention is predicated in part on the identification ofserum glycoproteins that are differentially glycosylated in subjectswith EAC, BE and in healthy patients. Accordingly, as described herein,detection of the levels of one or more of these different types ofglycosylation on particular glycoproteins (i.e., one or moreglycospecies) in a biological sample, such as a blood, serum or plasmasample, from a subject can be used to determine whether the subject hasEAC, BE or is healthy (i.e. has neither BE or EAC). The glycospecies canthus be considered biomarkers for BE and EAC.

In some instances, the present invention provides a method comprisingdetermining the ratio of a level of a glycospecies of a glycoprotein tothe total level of the glycoprotein in the sample, which glycospecies isdifferentially expressed between at least two of EAC, BE and HC, anddetermining a likelihood of the subject having or not having a HC basedon whether the ratio is above or below a predetermined threshold thatcorrelates with the presence of or absence of EAC or BE. Monitoring thelevels or ratios of one or more of the glycospecies identified hereincan also be used to monitor the progress of disease, such as before,during and/or after treatment. For example, levels or ratios of aparticular glycospecies identified herein as being increased ordecreased in subjects with EAC compared to a healthy control subject ora control subject with BE can be monitored during or after treatment. Achange in the level or ratio of one or more glycospecies in the subjectover time to be more similar to those levels or ratios observed incontrol subjects indicates that the disease has regressed. Conversely, achange over time in the level or ratio of one or more glycospecies inthe subject to be less similar to those levels or ratios observed incontrol subjects indicates that the disease has progressed. Methods ofmonitoring the disease progression of disease by assessing levels orratios of one or more glycospecies are thus also useful in assessing theefficacy of treatment, e.g. for assessing whether the treatment hasresulted in a regression of disease.

The glycoproteins identified herein as being differentially glycosylatedin subjects with EAC, BE and in healthy subjects are selected from:afamin, alpha-1-antichymotrypsin, alpha-1-antitrypsin, alpha-1-acidglycoprotein 1, alpha-1-acid glycoprotein 2, alpha-1B-glycoprotein,alpha-2-antiplasmin, alpha-2-HS-glycoprotein, alpha-2-macroglobulin,alpha-2-antiplasmin, angiotensinogen, antithrombin-III, apolipoproteinA-I, apolipoprotein B-100, .apolipoprotein M, beta-2-glycoprotein 1,C4b-binding protein alpha chain, C4b-binding protein beta chain,cadherin-5, centriolin, ceruloplasmin, coagulation factor XII,complement C1q subcomponent subunit B, complement C1q subcomponentsubunit C, complement C1s subcomponent, complement C5, complementcomponent C7, complement component C8 alpha chain, complement componentC9, complement factor B, complement factor H, complement factor I,fibrinogen gamma chain, fibronectin, ficolin-3, gelsolin, haptoglobin,hemoglobin subunit beta, hemopexin, histidine-rich glycoprotein,insulin-like growth factor-binding protein complex acid labile subunit,inter-alpha-trypsin inhibitor heavy chain H4, kallistatin, lumican,N-acetylmuramoyl-L-alanine amidase, plasma protease C1 inhibitor, plasmakallikrein, protein AMBP, prothrombin, retinol-binding protein 4,serotransferrin serum paraoxonase/arylesterase 1, serumparaoxonase/lactonase Sand vitamin K-dependent protein S.

The various types of glycosylation associated with the above serumglycoproteins exhibit different lectin-binding properties resulting fromthe different glycan structures on the proteins. These types ofglycosylation include, for example, glycosylations that bind to lectinshaving a general reactivity with α/β-D-Galactose, including Bauhiniapurpurea lectin (BPL, known to bind at least Galβ1-3GalNAc), Erythrinacristagalli agglutinin (ECA, known to bind at least Galβ1-4GlcNAc), andjacalin (JAC, known to bind at least Galα1-6GalNAc and Galβ1-3GalNAc),lectins having a general reactivity with D-N-Acetylgalactosamine,including soybean agglutinin (SBA, known to bind to at leastGalNAcα1-3Gal), Helix pomatia agglutinin (HPA, known to bind to at leasta-GalNAc), Wisteria floribunda agglutinin (WFA, known to bind to atleast GalNAcα1-6Gal and GalNAcα1-3GalNAc), Datura stramonium lectin(DSA, known to bind to at least β1-4GlcNAc oligomers), Helix aspersaagglutinin (HAA, known to bind to at least α-GlcNAc and α-GalNAc),Solanum tuberosum lectin (STL, known to bind to at leastGlcNAcβ1-4G1cNAc oligomers), and wheat germ agglutinin (WGA, known tobind to at least GlcNAcβ1-4GlcNAc and Neu5Ac), lectins having a generalreactivity with D-Mannose, including concanavalin A (ConA, known to bindto at least α-Man, α-Glc, and α-GlcNAc), Galanthus nivalis lectin (GNL,known to bind to at least Manα1-3Man) and Narcissus pseudonarcissus(NPL, known to bind to at least Manα1-6Man), lectins having a generalreactivity with α-L-Fucose, including Aleuria aurantia lectin (AAL,known to bind to at least Fucα1-2, -3, -6 linked), Pisum sativumagglutinin (PSA, known to bind to at least Fucα1-6GlcNAc of N-linkedglycans) and Ulex europeus agglutinin-I (UEA, known to bind to at leastFucα1-2Galβ1-4GlcNAc), lectins having a general reactivity with sialicacid, including Maackia amurensis agglutinin-II (MAA, known to bind toat least Neu5Acα2-3Galβ1-3 linkages) and Sambucus nigra agglutinin (SNA,known to bind to at least Neu5Acα2-6 linkages), and lectins having ageneral reactivity with complex specificities, includingerythroagglutinating phytohemagglutinin (EPHA, known to bind to at leastbisecting GlcNAc) and leukoagglutinating phytohemagglutinin (L-PHA,known to bind to at least tri/tetra-antennary β1-6GlcNAc).

Accordingly, the lectin-binding properties of the glycoproteins providedherein indicate the type of glycosylation. For example, an AAL-bindingglycoprotein, such as an AAL-binding glycospecies of gelsolin, is afucosylated type, possibly containing Fucαl-2, -3, -6 linkedoligosaccharides, while a NPL-binding glycoprotein, such as aNPL-binding glycospecies of gelsolin, is mannosylated, possiblycontaining Manα1-6Man oligosaccharides. The identity of the glycans onthe glycoprotein biomarkers described herein can be more preciselydetermined using standard methods well known in the art, such as, forexample, mass spectrometry, high-pressure liquid chromatography, nuclearmagnetic resonance, correlation spectroscopy, gas-liquid chromatography,or gas chromatography.

As described herein, the levels of the various types of glycosylationpresent on a single glycoprotein in a sample, such as a blood, serum orplasma sample, from subjects with

EAC is different to that of subjects with BE or healthy subjects.Similarly, the levels of several glycospecies of a single glycoproteinas listed above in the serum of subjects with BE is different to that ofsubjects with EAC or healthy subjects. Accordingly, the glycospeciesprovided herein are useful as biomarkers in methods for detecting andmonitoring the progress of EAC and BE, and related methods and uses.

2.1 Biomarkers that Distinguish subjects with EAC from Healthy Subjects

Among the glycospecies that are present at different levels in subjectswith EAC compared to healthy control subjects are glycospecies ofcomplement component C9, gelsolin, haptoglobin, complement factor B,alpha-1-antichymotrypsin, complement C5, hemopexin, C4b-binding proteinalpha chain, plasma protease C1 inhibitor, alpha-1-acid glycoprotein 1,alpha-1-acid glycoprotein 1, ceruloplasmin, antithrombin-III, ficolin-3,and complement C1q subcomponent subunit B. Other markers that arepresent at different levels in subjects with EAC compared to healthycontrol subjects are glycospecies of alpha-1-antitrypsin,alpha-1B-glycoprotein, complement C1s subcomponent, complement componentC7, alpha-1-acid glycoprotein 2, afamin, fibrinogen gamma chain,retinol-binding protein 4, gelsolin, vitamin K-dependent protein S,insulin-like growth factor-binding protein complex acid labile subunit,N-acetylmuramoyl-L-alanine amidase, fibrogen gamma chain,serotransferrin, haemoglobin subunit beta, haptoglobin, complementfactor B, alpha-2-HS-glycoprotein, cadherin-5, haemoglobin subunit beta,apolipoprotein A-I, fibrinogen beta chain, plasma kallikrein,kallistatin, cadherin-5, and centriolin.

Accordingly, a determination of the likelihood of whether a subject hasEAC or is healthy and does not have EAC can be made by assessing thelevel of one or more of these glycospecies in a biological sample fromthe subject, such as a serum, plasma or blood sample, and comparing itto the level of the same glycoprotein of the same glycosylation type ina corresponding sample from a healthy control subject (i.e. a subjectthat is known to not have EAC) or samples from multiple healthy controlsubjects, wherein an increase or decrease indicates that the subject hasEAC. In some instances, the level of the glycospecies is compared to apredetermined level or threshold, wherein an increase or decrease in thelevel of the subject compared to the threshold indicates that thesubject has EAC. The predetermined threshold may be calculated based onthe level of the same glycospecies in a corresponding sample from ahealthy control subject or from a group of healthy subjects, such that alevel of the glycospecies above or below the predetermined levelindicates that the subject has EAC. In some instances, the ratio of thelevel of one or more glycospecies to the total level of the glycoproteinis also increased or decreased in a subject with BE compared to ahealthy control subject or a group of healthy control subjects, and canthus also be used to determine the presence of EAC. Where two or moretypes of glycosylation are assessed for a single glycoprotein, aseparate ratio for each glycospecies can be determined. Alternatively, asingle ratio of the combined levels of the two or more types ofglycosylation with a single glycoprotein to the total level of theglycoprotein can be determined.

The glycospecies that are present at different levels in subjects withEAC compared to healthy control subjects are glycospecies of complementcomponent AAL-binding complement component C9, EPHA-binding complementcomponent C9, JAC-binding complement component C9, NPL-bindingcomplement component C9, PSA-binding complement component C9,WGA-binding complement component C9, AAL-binding gelsolin, EPHA-bindinggelsolin, JAC-binding gelsolin, PSA-binding gelsolin, EPHA-bindinghaptoglobin, NPL-binding haptoglobin, PSA-binding haptoglobin,WGA-binding haptoglobin, JAC-binding complement factor B, JAC-bindingalpha- 1-antichymotrypsin, NPL-binding alpha-1-antichymotrypsin,WGA-binding alpha-1-antichymotrypsin, JAC-binding complement C5,JAC-binding hemopexin, JAC-binding C4b-binding protein alpha chain,NPL-binding C4b-binding protein alpha chain, JAC-binding plasma proteaseC1 inhibitor, JAC-binding hemopexin, AAL-binding alpha-1-acidglycoprotein 1, EPHA-binding alpha-1-acid glycoprotein 1, JAC-bindingceruloplasmin, NPL-binding ceruloplasmin, NPL-binding antithrombin-III,STL-binding ficolin-3, WGA-binding complement C1q subcomponent subunitB.

Preferably, the individual glycospecies that are differentiallyexpressed between subjects with EAC and healthy subjects in a subjectare selected from the glycospecies identified in TABLE 1.

In other embodiments, the individual glycospecies that aredifferentially expressed between subjects with EAC and healthy subjectsin a subject are selected from the glycospecies identified in TABLE 2.

Accordingly, a determination of the likelihood of whether a subject hasEAC or is healthy (i.e., is not likely to have EAC) can be made byassessing the level of one or more glycospecies in a biological samplefrom the subject, such as a serum, plasma or blood sample, and comparingit to the level of the same one or more glycospecies in a correspondingsample from a healthy control subject (i.e. a subject that is known tonot have EAC) or samples from multiple healthy control subjects, whereinan increase or decrease indicates that the subject has EAC. In someinstances, the level of the one or more glycospecies is compared to apredetermined level or threshold, wherein an increase or decrease in thelevel of the subject compared to the threshold indicates that thesubject has EAC. The predetermined threshold may be calculated based onthe level of the same glycospecies in a corresponding sample from ahealthy control subject or from a group of healthy subjects, such that alevel of the glycospecies above or below the predetermined levelindicates that the subject has EAC. In some instances, the ratio of thelevel of a glycospecies to the total level of the correspondingglycoprotein is also increased or decreased in a subject with EACcompared to a healthy control subject or a group of healthy controlsubjects, and can thus also be used to determine the likelihood of thepresence of

EAC. Where two or more glycospecies are assessed for a singleglycoprotein, a separate ratio for each glycospecies can be determined.Alternatively, a single ratio of the combined levels of the glycospeciesof the single glycoprotein to the total level of the glycoprotein can bedetermined.

Illustrative glycospecies that have increased levels in subjects withEAC compared to healthy subjects include, for example, thoseglycospecies identified in TABLE 1 as being overexpressed in subjectswith EAC. Thus, a determination that a subject has an increased level ofone or more of these types of glycosylation compared to a healthycontrol subject or compared to a predetermined threshold indicates thatthe subject has EAC. Similarly, a determination that a subject has anincreased ratio of the level of a glycoprotein with one or more types ofglycosylation to the total level of the glycoprotein compared to theratio in a healthy control subject or compared to a predeterminedthreshold indicates that the subject has EAC.

Illustrative glycospecies that have decreased levels in subjects withEAC compared to healthy subjects include, for example, thoseglycospecies identified as being underexpressed in subjects with EAC.Thus, a determination that a subject has a decreased level of one ormore of these glycospecies compared to a healthy control subject orcompared to a predetermined threshold indicates that the subject hasEAC. Similarly, a determination that a subject has a decreased ratio ofthe level of one or more of these glycospecies to the total level of theglycoprotein compared to the ratio in a healthy control subject orcompared to a predetermined threshold indicates that the subject hasEAC.

The levels or ratios of the glycospecies identified above as beinguseful biomarkers for EAC can also be used to monitor the progress ofdisease in a subject that has

EAC. For example, the progress of EAC can be assessed or monitoredbefore, during or after treatment by assessing the level or ratio (i.e.the ratio of the level of a glycospecies to the total level of theglycoprotein) of one of more glycospecies in samples taken at varioustime points. Accordingly, the efficacy of treatment can also be assessedby determining the level or ratio of one or more glycospecies in samplestaken at various time points, wherein at least one of those time pointsis during or after treatment. An increase over time in the level orratio of one or more of the glycospecies identified above as beingincreased in subjects with EAC compared to healthy subjects indicatesthat the disease has progressed, while a decrease in one or more ofthese glycospecies indicates that the disease has regressed. Conversely,a decrease over time in the level or ratio of one or more of theglycospecies identified above as being decreased in subjects with EACcompared to healthy subjects indicates that the disease has progressed,while an increase in one or more of these glycospecies indicates thatthe disease has regressed. In instances where the subject has undergoneor is undergoing treatment for EAC, progression of the disease mayindicate that such treatment has not been effective, while regression ofthe disease may indicate that such treatment has been at least partiallyeffective.

Accordingly, an increase over time in the level or ratio of one or moreof the glycospecies identified in TABLE 1, TABLE 2, or TABLE 3 as beingoverexpressed in those subjects which are diagnosed with EAC indicatesthat the subject's EAC has progressed over time, while a decrease of anyone or more of these glycospecies indicates that the subject's EAC hasregressed.

A decrease over time in the level or ratio of one or more of theglycospecies identified in TABLE 1, TABLE 2, or TABLE 3 as beingunderexpressed in those subjects diagnosed with EAC indicates that thesubject's EAC has progressed over time, while an increase of any one ormore of these glycospecies indicates that the subject's EAC hasregressed.

In some instances, the level or ratio of more than one type ofglycosylation with a single glycoprotein identified above is assessed todetermine the presence or progression of EAC in a subject. For example,2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more types of glycosylation with asingle glycoprotein may be assessed. Thus, a panel of more than oneglycospecies may be assessed to determine a glycoprotein's glycosylationprofile or signature for a subject. In some embodiments, this profilecan be compared to a corresponding profile from a control subject or agroup of control subjects to determine the presence or progression ofEAC, wherein a change in the profile resulting from increases ordecreases in the levels or ratios of the various types of glycosylationfor a single glycoprotein, as described above, indicates the presence orprogression of EAC.

2.2 Biomarkers that Distinguish Subjects with BE from Healthy Subjects

Among the glycospecies that are present at different levels in subjectswith BE compared to healthy control subjects are glycospecies ofalpha-2-macroglobulin, apolipoprotein B-100, ficolin-3, complement C1qsubcomponent subunit C, protein AMBP, alpha-1-acid glycoprotein 1,coagulation factor XII, antithrombin-III, alpha-2-antiplasmin, andceruloplasmin.

Additional glycospecies that are present at different levels in subjectswith BE compared to healthy control subjects are glycospecies oflumican, prothrombin, fibronectin, apolipoprotein M, angiotensinogen,apolipoprotein A-I, fibrinogen beta chain, plasma kallikrein,histidine-rich glycoprotein, vitamin K-dependent protein S, complementcomponent C8 alpha chain, complement factor H, kallistatin, afamin, andhemoglobin subunit beta.

Accordingly, a determination of whether a subject has BE or is healthyand does not have BE can be made by assessing the level of one or moreof these glycospecies in a biological sample from the subject, such as aserum, plasma or blood sample, and comparing it to the level of the sameglycoprotein of the same glycosylation type in a corresponding samplefrom a healthy control subject (i.e. a subject that is known to not haveBE) or samples from multiple healthy control subjects, wherein anincrease or decrease indicates that the subject has BE. In someinstances, the level of the glycospecies is compared to a predeterminedlevel or threshold, wherein an increase or decrease in the level of thesubject compared to the threshold indicates that the subject has BE. Thepredetermined threshold may be calculated based on the level of the sameglycospecies in a corresponding sample from a healthy control subject orfrom a group of healthy subjects, such that a level of the glycospeciesabove or below the predetermined level indicates that the subject hasBE. In some instances, the ratio of the level of one or moreglycospecies to the total level of the glycoprotein is also increased ordecreased in a subject with BE compared to a healthy control subject ora group of healthy control subjects, and can thus also be used todetermine the presence of BE. Where two or more types of glycosylationare assessed for a single glycoprotein, a separate ratio for eachglycospecies can be determined. Alternatively, a single ratio of thecombined levels of the two or more types of glycosylation with a singleglycoprotein to the total level of the glycoprotein can be determined.

The glycospecies that are differentially expressed between healthysubjects and those diagnosed with BE are selected from the groupcomprising or consisting of: EPHA-binding alpha-2-macroglobulin,JAC-binding apolipoprotein B-100, NPL-binding apolipoprotein B-100,AAL-binding ficolin-3, STL-binding ficolin-3, AAL-binding complement C1qsubcomponent subunit C, EPHA-binding protein AMBP, EPHA-bindingalpha-1-acid glycoprotein 1, JAC-binding coagulation factor XII,NPL-binding antithrombin-III, SNA-binding alpha-2-antiplasmin, andSTL-binding ceruloplasmin

Preferably, the individual glycospecies (i.e., defined by theglycan-binding molecule (e.g., lectin) and the glycoprotein to which itbinds) that are differentially expressed between subjects with BE andhealthy subjects are selected from TABLE 4.Thus, a determination that asubject has an increased level of one or more of those glycospeciesidentified in TABLE 4 or TABLE 5 as being overexpressed as compared to ahealthy control subject or compared to a predetermined thresholdindicates that the subject has BE. Similarly, a determination that asubject has an increased ratio of the level of one or more of theseglycospecies to the total level of the corresponding glycoproteincompared to the ratio in a healthy control subject or compared to apredetermined threshold indicates that the subject has BE.

Illustrative glycospecies that have decreased levels in subjects with BEcompared to healthy subjects include, for example, those glycospeciesidentified in TABLE 4 or TABLE 5 as being underexpressed in subjectsdiagnosed with BE. Thus, a determination that a subject has a decreasedlevel of one or more of these glycospecies compared to a healthy controlsubject or compared to a predetermined threshold indicates that thesubject has BE. Similarly, a determination that a subject has adecreased ratio of the level of one or more of those glycospecies to thetotal level of the corresponding glycoprotein compared to the ratio in ahealthy control subject or compared to a predetermined thresholdindicates that the subject has

BE.

The levels or ratios of the glycospecies of one or more glycoproteinsidentified above as being useful biomarkers for BE can also be used tomonitor the progress of disease in a subject that has BE. For example,the progress of BE can be assessed or monitored before, during or aftertreatment by assessing the level or ratio (i.e. the ratio of the levelof a glycospecies to the total level of the glycoprotein) of one of moreof the glycospecies in samples taken at various time points.Accordingly, the efficacy of treatment can also be assessed bydetermining the level or ratio of one of more of the glycospecies insamples taken at various time points, wherein at least one of those timepoints is during or after treatment. An increase over time in the levelor ratio of one or more of the glycospecies identified above as beingincreased in subjects with BE compared to healthy subjects indicatesthat the disease has progressed, while a decrease in one or more ofthese glycospecies indicates that the disease has regressed.

Conversely, a decrease over time in the level or ratio of one or more ofthe glycospecies identified above as being decreased in subjects with BEcompared to healthy subjects indicates that the disease has progressed,while an increase in one or more of these glycospecies indicates thatthe disease has regressed. In instances where the subject has undergoneor is undergoing treatment for BE, progression of the disease mayindicate that such treatment has not been effective, while regression ofthe disease may indicate that such treatment has been at least partiallyeffective.

Accordingly, an increase over time in the level or ratio of one or moreof those glycospecies identified in TABLE 4 or TABLE 5 as beingoverexpressed in a subject diagnosed with BE indicates that thesubject's BE has progressed over time, while a decrease of any one ormore of these glycospecies indicates that the subject's BE hasregressed.

A decrease over time in the level or ratio of alpha-2-macroglobulin in asubject with BE indicates that the subject's BE has progressed overtime, while an increase of this glycospecies indicates that thesubject's BE has regressed.

In some instances, the level or ratio of more than one glycospeciesidentified above is assessed to determine the presence or progression ofBE in a subject. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or moreglycospecies may be assessed. Thus, a panel of glycospecies may beassessed to determine a glycospecies profile or signature for a subject.In some embodiments, this profile can be compared to a correspondingprofile from a control subject or a group of control subjects todetermine the presence or progression of BE, wherein a change in theprofile resulting from increases or decreases in the levels or ratios ofthe various glycospecies, as described above, indicates the presence orprogression of BE.

2.3 Biomarkers that Distinguish Subjects with EAC from Subjects with BE

Among the glycospecies that are present at different levels in abiological sample from a subject with EAC compared to subjects with BEare glycospecies of haptoglobin, alpha- 1-antichymotrypsin, complementcomponent C9, complement factor B, complement C5, hemopexin, C4b-bindingprotein alpha chain, plasma protease C1 inhibitor, gelsolin,alpha-2-macroglobulin, alpha-2-HS-glycoprotein, ficolin-3, complementC1q subcomponent subunit C, alpha-1-acid glycoprotein 1, ceruloplasmin,coagulation factor XII, and alpha-2-antiplasmin Additional glycospeciesthat are present at different levels in a biological sample from asubject with EAC compared to subjects with BE are glycospecies ofprothrombin, hemopexin, alpha-1B-glycoprotein, complement factor I,complement factor H, complement Cls subcomponent, C4b-binding proteinbeta chain, Inter-alpha-trypsin inhibitor heavy chain H4,retinol-binding protein 4, beta-2-glycoprotein 1, lumican, and serumparaoxonase/lactonase 3.

Accordingly, a determination of whether a subject has EAC can be made byassessing the level of one or more of these glycospecies in a biologicalsample, such as a serum, plasma or blood sample, from the subject, andcomparing it to the level of the same glycospecies in a correspondingsample from a control subject with BE or from multiple control subjects,wherein an increase or decrease indicates that the subject has EAC. Insome instances, the abundance or level of the glycospecies is comparedto a predetermined threshold, wherein an increase or decrease indicatesthat the subject has EAC. The predetermined threshold is determinedbased on the level of the same glycospecies in a corresponding samplefrom a control subject with BE or from a group of control subjects withBE, such that a level of the glycospecies in the sample from the subjectabove or below the predetermined level indicates that the subject hasEAC. In some instances, the ratio of the level of one or moreglycospecies of a glycoprotein to the total level of the glycoprotein isalso increased or decreased in a subject with EAC compared to a controlsubject with BE or a group of control subjects with BE. Where two ormore glycospecies of the same glycoprotein are assessed, a separateratio for each glycospecies can be determined. Alternatively, a singleratio of the combined levels of the two or more glycospecies of theglycoprotein to the total level of the glycoprotein can be determined.

Among the glycospecies that are present at different levels in subjectswith EAC compared to subjects with BE are glycospecies of AAL-bindingcomplement component C9, EPHA-binding complement component C9,WGA-binding complement component C9, JAC-binding complement componentC9, NPL-binding complement component C9, PSA-binding complementcomponent C9, AAL-binding gelsolin, EPHA-binding gelsolin, JAC-bindinggelsolin, PSA-binding gelsolin, NPL-binding gelsolin, WGA-bindinggelsolin, AAL-binding haptoglobin, EPHA-binding haptoglobin, JAC-bindinghaptoglobin, PSA-binding haptoglobin, WGA-binding haptoglobin,JAC-binding complement factor B, EPHA-binding alpha-1-antichymotrypsin,PSA-binding alpha-1 -antichymotrypsin, JAC-bindingalpha-1-antichymotrypsin, AAL-binding complement CS, JAC-bindingcomplement CS, PSA-binding complement CS, AAL-binding complementcomponent C7, PSA-binding complement component C7, EPHA-bindingcomplement component C7, JAC-binding complement component C7,AAL-binding apolipoprotein B-100, NPL-binding apolipoprotein B-100,EPHA-binding serotransferrin, JAC-binding alpha-1 -antitrypsin,JAC-binding alpha-1B-glycoprotein, AAL-binding alpha-1-acid glycoprotein1, AAL-binding ficolin-3, AAL-binding complement C1q subcomponentsubunit C, AAL-binding alpha-1-acid glycoprotein 1, JAC-bindingceruloplasmin, STL-binding ceruloplasmin, JAC-binding coagulation factorXII, and SNA-binding alpha-2-antiplasmin

Preferably, the individual glycospecies (i.e., defined by theglycan-binding molecule (e.g., lectin) and the glycoprotein to which itbinds) that are differentially expressed between EAC and BE are selectedfrom TABLE 6.

In other embodiments, the individual glycospecies (i.e., defined by theglycan-binding molecule (e.g., lectin) and the glycoprotein to which itbinds) that are differentially expressed between EAC and BE are selectedfrom TABLE 7.

Thus, a determination that a subject has a decreased level of one ormore of these glycospecies compared to a control subject with BE orcompared to a predetermined threshold indicates that the subject hasEAC. Similarly, a determination that a subject has a decreased ratio ofthe level of one or more of these glycospecies of a glycoprotein to thetotal level of the glycoprotein compared to the ratio in a controlsubject with BE or compared to a predetermined threshold indicates thatthe subject has EAC.

In particular examples, the levels of different glycospecies of gelsolinare assessed to determine whether a subject has EAC. As indicated above,the levels of multiple glycospecies of gelsolin are reduced in EACsubjects compared to BE subjects, as set out in TABLE 6 or TABLE 7.These isoforms of gelsolin include AAL-binding gelsolin, JAC-bindinggelsolin, and PSA-binding gelsolin, and comparing the level to the levelof the same type of glycosylation of gelsolin in a control subject withBE or to a predetermined threshold, wherein a decrease indicates thatthe subject has EAC.

In further examples, the ratio of the level of one or more glycospeciesof gelsolin to the total gelsolin level in a sample from a subject isassessed to determine whether the subject has EAC. Thus, in someexamples, the methods of the present invention include determining thelevel of a gelsolin glycospecies selected from the group consisting ofAAL-binding gelsolin, JAC-binding gelsolin and PSA-binding gelsolin, ina sample from a subject and also determining the total gelsolin levelsin the same sample. A ratio of the level of the gelsolin with a singletype of glycosylation to the total gelsolin level is then determined andcompared to the same ratio as determined for a control subject with BEor is compared to a predetermined threshold. A decrease in the ratio inthe subject compared to the control subject or predetermined thresholdindicates that the subject has EAC. In some instances, the level of twoor more types of glycosylation of gelsolin, such as two or more ofAAL-binding gelsolin, JAC-binding gelsolin and PSA-binding gelsolin, aremeasured so as to determine a ratio of the combined level of the two ormore types of glycosylation of gelsolin to the total level of gelsolin.This ratio is then compared to the same ratio as determined for acontrol subject with BE or is compared to a predetermined threshold,wherein a decrease in the ratio in the subject compared to the controlsubject or predetermined threshold indicates that the subject has EAC.

The levels or ratios of the glycospecies identified above as beinguseful biomarkers for EAC can also be used to monitor the progress ofdisease in a subject that has EAC. For example, the progress of EAC canbe assessed or monitored before, during or after treatment by assessingthe level or ratio (i.e. the ratio of the level of a glycospecies of aglycoprotein to the total level of the glycoprotein) of one of more ofthe glycospecies in samples taken at various time points. Accordingly,the efficacy of treatment can also be assessed by determining the levelor ratio of one of more of the glycospecies in samples taken at varioustime points, wherein at least one of those time points is during orafter treatment. An increase over time in the level or ratio of one ormore of the glycospecies identified above as being increased in subjectswith EAC compared to BE or a healthy sample indicates that the diseasehas progressed, while a decrease in one or more of these glycospeciesindicates that the disease has regressed. Conversely, a decrease overtime in the level or ratio of one or more of the glycospecies identifiedabove as being decreased in subjects with EAC compared to BE or ahealthy sample indicates that the disease has progressed, while anincrease in one or more of these glycospecies indicates that the diseasehas regressed. In instances where the subject has undergone or isundergoing treatment for EAC, progression of the disease may indicatethat such treatment has not been effective, while regression of thedisease may indicate that such treatment has been at least partiallyeffective.

Therefore, an increase over time in the level or ratio of one or more ofglycospecies of glycoprotein selected from those identified in TABLE 6or TABLE 7 as being overexpressed in a subject with EAC compared with asubject with BE, in a subject diagnosed with EAC indicates that thesubject's EAC has progressed over time, while a decrease of any one ormore of these glycospecies indicates that the subject's EAC hasregressed.

A decrease over time in the level or ratio of one or more thoseglycospecies of glycoprotein that are identified in TABLE 6 or TABLE 7as being underexpressed in a subject with EAC compared to a subject withBE, in a subject diagnosed with EAC indicates that the subject's EAC hasprogressed over time, while an increase of any one or more of theseglycospecies indicates that the subject's EAC has regressed.

In some instances, the level or ratio of more than one glycospeciesidentified above is assessed to determine the presence or progression ofEAC in a subject. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 ormore glycospecies may be assessed. Thus, a panel of glycospecies may beassessed to determine a “glycoprotein profile” for a subject. In someembodiments, this profile can be compared to a corresponding profilefrom a control subject or a group of control subjects to determine thepresence or progression of EAC, wherein a change in the profileresulting from increases or decreases in the levels or ratios of thevarious glycospecies, as described above, indicates the presence orprogression of EAC.

3. Methods for Assessing the Levels of Biomarkers

The levels of glycospecies identified herein as being useful biomarkersfor detecting the likelihood of the presence or absence of, ormonitoring the progress of, EAC and BE, can be assessed by any methodknown in the art. Such methods include, but are not limited to, methodsthat detect binding of a glycospecies to a glycan-binding molecule, suchas a lectin, glycospecific antibody or glycospecific aptamer that isselective and/or specific for the glycospecies, such as western blots,ELISAs and microarray-based techniques. Spectroscopic methods also canbe used to assess the level of a glycospecies in a sample.

The level of one or more glycospecies as described herein is assessed ina biological sample from a subject. Most typically, the biologicalsample is a blood, serum, plasma or blood fraction sample, althoughother types of samples are contemplated. The sample may be obtained fromthe subject before or after diagnosis of BE or EAC. For example, in themethods of the present invention that are used to detect BE or EAC, thesample may be obtained from a subject before or after that subject hasbeen diagnosed with having or having had BE or EAC. In the methods ofthe present invention that are used to monitor the progress of disease,the sample is obtained from the subject after they have been diagnosedwith having or having had BE or EAC. For example, in some instances, thesubject has been diagnosed with BE or EAC, then been assessed as havingcleared the disease before the sample is taken to assess the levels ofone or more glycospecies. In instances where the subject has beendiagnosed as having or having had BE or EAC, the subject may haveundergone or be undergoing treatment, such as surgical or medicaltreatment. One or more samples can be taken from the subject at one ormore time points. For example, to monitor the progress of disease, atleast two samples are taken at two different time points, so as tocompare the levels of one or more glycoproteins over time. In particularexamples, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more samples are taken from thesubject over a period of days, weeks, months or years.

In particular embodiments of the methods of the invention, the level ofa glycospecies is assessed by detecting the binding of the glycospeciesto an appropriate glycan-binding molecule. In one example, theglycan-binding molecule is a lectin. Lectins are proteins orglycoproteins that bind to all or part of a glycan structure. A lectinmay bind to a specific glycan moiety that is part of a glycoprotein oranother glycan-containing molecule such as a glycolipid,glycophosphatidylinositol or glycosaminoglycan. Lectins are capable ofbinding to specific glycans. Advantageously, the high specificity of alectin for a particular glycan moiety facilitates the precipitation,isolation and/or detection of glycoproteins with a particular singletypes of glycosylation from or in a biological sample by specificallybinding to those types of glycosylation.

Lectins useful for binding glycospecies of glycoproteinare describedherein so as to determine the levels of the glycospecies in a sampleinclude Bauhinia purpurea lectin (BPL), Erythrina cristagalli agglutinin(ECA), jacalin (JAC), soybean agglutinin (SBA), Helix pomatia agglutinin(HPA), Wisteria floribunda agglutinin (WFA), Datura stramonium lectin(DSA), Helix aspersa agglutinin (HAA), Solanum tuberosum lectin (STL),wheat germ agglutinin (WGA), concanavalin A (ConA), Galanthus nivalislectin (GNL), Narcissus pseudonarcissus lectin (NPL), Aleuria aurantialectin (AAL), Pisum sativum agglutinin (PSA), Ulex europeus agglutinin-I(UEA), Maackia amurensis agglutinin-II (MAA), Sambucus nigra agglutinin(SNA), erythroagglutinating phytohemagglutinin (E-PHA), andleukoagglutinating phytohemagglutinin (L-PHA). For example, AAL can beused to detect the levels of any AAL-binding glycoprotein, such asAAL-gelsolin and AAL-binding complement component C9. Similarly, PSA canbe used to detect the levels of any PSA-binding glycoprotein, including,but not limited to, PSA-binding complement CS and PSA-bindingretinol-binding protein 4.

Assays and techniques to detect binding of a glycospecies to a lectinare well known in the art, and any such assay or technique can be usedin the methods of the present invention. In one example, lectin-magneticbead array-coupled mass spectrometry (LeMBA-MS), as described by Choi etal. (Electrophoresis (2011) 32, 3564-3575) and also below in Example 1,is used. In other examples, immunoassays that utilise lectin to capturethe glycoprotein with a single glycosylation and a glycoprotein-specificantibody to detect and quantify the captured glycoprotein are used. Suchassays include, for example, western blots, ELISAs, lectin-AlphaLISAassays, and immunofluorescence. For example, lectin-based ELISAs (orlectin-based immunosorbant assays (LIAs)) may involve coating a surface,such as a multiwell plate, with an antibody specific for the polypeptidebackbone of the glycoprotein, then adding the biological samplecontaining the glycoprotein to form an immobilised complex. The complexis then contacted with the appropriate biotinylated lectin and detectedusing streptavidin. In other example, the plate is coated with lectinand the biological sample is added to form lectin-glycoproteincomplexes, which are then detected using antibodies specific for thepolypeptide backbone of the glycoprotein. These types of techniques areamenable to modification for use in clinical and diagnostic applicationsthat require high sensitivity and accuracy with relatively low cost. Forexample, automated analyzers based on liquid-phase binding can be usedto detect and quantify specific glycoproteins in a biological sample,such as a serum sample. Choi et al. (Clinica Chimica Acta (2012)413:170-174) and Kagebayashi et al. (Anal Biochem (2009) 388:306-311)describe the use of the micro-total analysis systems (μTAS)immunoassays, performed using fully automated instruments such as theμTASWako® i30 Immunoanalyzer (Wako Pure Chemicals Industries, Ltd) todetect and quantify an LCA-binding glycoprotein of alpha-fetoprotein,and such systems are readily adaptable to detect and quantify theglycoproteins described herein.

Techniques involving the use of electrochemical sensors are alsosuitable for use in the methods of the invention to assess levels of aglycospecies in a sample by binding the glycospecies to a lectin.Electrochemical sensors include a biorecognition element, such as alectin, coupled to an electrode transducer surface. The specificinteraction of a biological sample containing, for example, aglycospecies, to its corresponding lectin on the electrode surface isdetected by electrical current or potential changes occurring at thetransducer/biomolecule interface. This type of technique is described byShah, A. K., Electrochemical detection of glycan and protein epitopes ofglycoproteins in serum, Analyst, 2014, 139(22): 5970-6, and isparticularly suited to point of care applications.

Thus, the present invention is also directed to the use of lectins andcompositions comprising lectins to detect the levels of one or moreglycospecies in a biological sample, and thus uses of lectins todetermine the likelihood of the presence or absence of, or monitor theprogress of, EAC or BE in subject. For example, provided are uses of alectin to determine the level of one or more glycospecies in a sample,comprising contacting the sample with the lectin under conditions thatpermit binding of the glycospecies to the lectin, and detecting anddetermining the level of the glycospecies in the sample, wherein a levelof the glycospecies above or below a predetermined threshold indicatesthe presence of EAC or BE, as described above in section 2. Similarly,lectins can be used to determine the ratio of the level of aglycospecies in a sample to the total level of the glycoprotein, asdescribed above, wherein a ratio of the glycospecies above or below apredetermined threshold indicates the presence of EAC or BE, asdescribed above in section 2.

In some of the methods of the present invention, a ratio of the level ofa glycospecies to the total level of the glycoprotein (i.e. the combinedlevel of all types or glycosylation of the glycoprotein) in a sample isdetermined. Thus, the methods of the present invention may also requireassessing the total level of a glycoprotein in a sample. Any method fordetermining the level of a glycoprotein, such as the concentration oramount of a glycoprotein, in a samples can be used, and such methods arewidely known to those of skill in the art. Exemplary methods include,but are not limited to, immunoassays that utilise glycoprotein-specificantibodies to capture the glycoproteins and a secondary antibody todetect and quantify the captured glycoprotein. Such assays include, forexample, western blots, ELISAs and immunofluorescence. The μTASimmunoassays described above are also suitable for detecting andquantifying levels of a glycoprotein, as are techniques involving theuse of electrochemical sensors, as described above. In some embodiments,the detection and quantitation of the glycoprotein is performedsimultaneously with, and using the same platform, as detection andquantitation of the glycospecies. For example, the levels of aparticular glycospecies and the total levels of the glycoprotein can besimultaneously assessed using the μTAS immunoassays referred to aboveand described by Choi et al. (Clinica Chimica Acta (2012) 413:170-174)and Kagebayashi et al. (Anal Biochem (2009) 388:306-311).

A glycospecies may be determined to be a multivariate marker, i.e.,differentially expressed in a variety of ways, for example, betweensubjects or group of subjects with different conditions if the presenceor absence or mean or median level or concentration of the glycospeciesin the different subjects or group of subjects is calculated to bestatistically significant. Common tests for statistical significanceinclude, among others, t-test, ANOVA, Kruskal-Wallis, Wilcoxon,Mann-Whitney and odds ratio.

4. Kits

All of the essential materials and reagents required for detecting anddetermining the levels of one or more glycospecies described herein maybe assembled together in a kit. The kits may also optionally includeappropriate reagents for detection of labels, positive and negativecontrols, washing solutions, blotting membranes, microtiter platesdilution buffers and the like. For example, a lectin-bindingimmunosorbent assay may include a lectin specific for the glycospeciesof the glycoprotein to be detected, an antibody specific for theglycoprotein (i.e. specific for the polypeptide backbone of theglycosylation), and optionally a glycospecies, which may be used as apositive control. Also included may be buffers, wash solutions orblocking reagents, and enzymes and/or substrates for detection oflabels. The kit can also feature various devices and reagents forperforming one of the assays described herein, and/or printedinstructions for using the kit to determine the level of a glycoprotein.

5. Therapeutic Applications

The present invention also extends to the management and treatment ofsubjects with BE or EAC. For example, where the methods of the presentinvention are used to detect the presence of EAC or BE in a subject, themethods can further comprise treating the EAC or BE. Therapies for EACand BE are well known in the art and an appropriate therapeutic regimenfor a particular subject, based on the severity of the disease and otherfactors, such as age and general health of the subject, can bedetermined by a skilled practitioner and administered appropriatelywithout undue experimentation.

Treatment for BE may include, for example, changes to diet and exercise,administration of therapeutic agents to reduce acid reflux, includingproton pump inhibitors, antacids or H2 blockers, photodynamic therapy(PDT) and endoscopic mucosal resection (EMR). Treatment options for EACcan vary depending on the stage of the cancer, i.e. stage 1, 2, 3, 4 or5, and can include surgery to remove the part of their esophagus thatcontains the cancer (esophagectomy), chemotherapy, immunotherapiesand/or radiation therapy.

Radiotherapies include radiation and waves that induce DNA damage forexample, γ-irradiation, X-rays, UV irradiation, microwaves, electronicemissions, radioisotopes, and the like. Therapy may be achieved byirradiating the localized tumor site with the above described forms ofradiations. It is most likely that all of these factors effect a broadrange of damage DNA, on the precursors of DNA, the replication andrepair of DNA, and the assembly and maintenance of chromosomes.

Dosage ranges for X-rays range from daily doses of 50 to 200 roentgensfor prolonged periods of time (3 to 4 weeks), to single doses of 2000 to6000 roentgens. Dosage ranges for radioisotopes vary widely, and dependon the half life of the isotope, the strength and type of radiationemitted, and the uptake by the neoplastic cells.

Non-limiting examples of radiotherapies include conformal external beamradiotherapy (50-100 Grey given as fractions over 4-8 weeks), eithersingle shot or fractionated, high dose rate brachytherapy, permanentinterstitial brachytherapy, systemic radio-isotopes (e.g., Strontium89). In some embodiments the radiotherapy may be administered incombination with a radiosensitizing agent. Illustrative examples ofradiosensitizing agents include but are not limited to efaproxiral,etanidazole, fluosol, misonidazole, nimorazole, temoporfin andtirapazamine.

Chemotherapeutic agents may be selected from any one or more of thefollowing categories:

(i) antiproliferative/antineoplastic drugs and combinations thereof, asused in medical oncology, such as alkylating agents (for examplecis-platin, carboplatin, cyclophosphamide, nitrogen mustard, melphalan,chlorambucil, busulphan and nitrosoureas), antimetabolites (for exampleantifolates such as fluoropyridines like 5-fluorouracil and tegafur,raltitrexed, methotrexate, cytosine arabinoside and hydroxyurea,anti-tumor antibiotics (for example anthracyclines like adriamycin,bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C,dactinomycin and mithramycin), antimitotic agents (for example vincaalkaloids like vincristine, vinblastine, vindesine and vinorelbine andtaxoids like paclitaxel and docetaxel, and topoisomerase inhibitors (forexample epipodophyllotoxins like etoposide and teniposide, amsacrine,topotecan and camptothecin);

(ii) cytostatic agents such as antiestrogens (for example tamoxifen,toremifene, raloxifene, droloxifene and idoxifene), oestrogen receptordown regulators (for example fulvestrant), antiandrogens (for examplebicalutamide, flutamide, nilutamide and cyproterone acetate), UHantagonists or LHRH agonists (for example goserelin, leuprorelin andbuserelin), progestogens (for example megestrol acetate), aromataseinhibitors (for example as anastrozole, letrozole, vorozole andexemestane) and inhibitors of 5α-reductase such as finasteride;

(iii) agents which inhibit cancer cell invasion (for examplemetalloproteinase inhibitors like marimastat and inhibitors of urokinaseplasminogen activator receptor function);

(iv) inhibitors of growth factor function, for example such inhibitorsinclude growth factor antibodies, growth factor receptor antibodies (forexample the anti-erbb2 antibody trastuzumab [Herceptin™] and theanti-erbb1 antibody cetuximab [C225]), farnesyl transferase inhibitors,MEK inhibitors, tyrosine kinase inhibitors and serine/threonine kinaseinhibitors, for example other inhibitors of the epidermal growth factorfamily (for example other EGFR family tyrosine kinase inhibitors such asN-(3-chloro-4-fluorophenyl)-7-methoxy-6-(3-morpholinopropoxy)quinazolin-4-amine(gefitinib, AZD1839),N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine(erlotinib, OSI-774) and6-acrylamido-N-(3-chloro-4-fluorophenyl)-7-(3-morpholinopropoxy)quinazoli-n-4-amine (CI 1033)), for example inhibitors of the platelet-derivedgrowth factor family and for example inhibitors of the hepatocyte growthfactor family;

(v) anti-angiogenic agents such as those which inhibit the effects ofvascular endothelial growth factor, (for example the anti-vascularendothelial cell growth factor antibody bevacizumab [Avastin™],compounds such as those disclosed in International Patent ApplicationsWO 97/22596, WO 97/30035, WO 97/32856 and WO 98/13354) and compoundsthat work by other mechanisms (for example linomide, inhibitors ofintegrin αvβ33 function and angiostatin);

(vi) vascular damaging agents such as Combretastatin A4 and compoundsdisclosed in International Patent Applications WO 99/02166, W000/40529,WO 00/41669, W001/92224, W002/04434 and W002/08213;

(vii) antisense therapies, for example those which are directed to thetargets listed above, such as ISIS 2503, an anti-ras antisense, and

(viii) gene therapy approaches, including for example approaches toreplace aberrant genes such as aberrant p53 or aberrant GDEPT(gene-directed enzyme pro-drug therapy) approaches such as those usingcytosine deaminase, thymidine kinase or a bacterial nitroreductaseenzyme and approaches to increase patient tolerance to chemotherapy orradiotherapy such as multi-drug resistance gene therapy.

Immunotherapy approaches, include for example ex-vivo and in-vivoapproaches to increase the immunogenicity of patient tumor cells, suchas transfection with cytokines such as interleukin 2, interleukin 4 orgranulocyte-macrophage colony stimulating factor, approaches to decreaseT-cell anergy, approaches using transfected immune cells such ascytokine-transfected dendritic cells, approaches usingcytokine-transfected tumor cell lines and approaches usinganti-idiotypic antibodies. These approaches generally rely on the use ofimmune effector cells and molecules to target and destroy cancer cells.The immune effector may be, for example, an antibody specific for somemarker on the surface of a malignant cell. The antibody alone may serveas an effector of therapy or it may recruit other cells to actuallyfacilitate cell killing. The antibody also may be conjugated to a drugor toxin (chemotherapeutic, radionuclide, ricin A chain, cholera toxin,pertussis toxin, etc.) and serve merely as a targeting agent.Alternatively, the effector may be a lymphocyte carrying a surfacemolecule that interacts, either directly or indirectly, with a malignantcell target. Various effector cells include cytotoxic T cells and NKcells.

Examples of other cancer therapies include phototherapy, cryotherapy,toxin therapy or pro-apoptosis therapy. One of skill in the art wouldknow that this list is not exhaustive of the types of treatmentmodalities available for cancer and other hyperplastic lesions.

In instances where the cancer is HER2-positive, treatment may alsoinclude administration of an anti-HER2 antibody, such as trastuzumab.

Where the methods of the present invention are used to monitor theprogress of EAC or BE in a subject that has undergone or is undergoingtreatment, and/or assess the efficacy of treatment, the methods may alsoinclude modifying or altering the treatment. For example, if the levelor ratio of one or more glycospecies identified herein indicates thatthe disease has progressed and the current or previous treatmentprotocol has been ineffective, a skilled practitioner may devise amodified or altered treatment protocol. For example, if the subject hasundergone surgery to remove part of the esophagus, and the level orratio of one or more glycospecies identified herein indicates that thedisease has progressed post-surgery, the subject may be administeredchemotherapy and/or radiotherapy. Conversely, if the level or ratio ofone or more glycospecies identified herein indicates that the diseasehas regressed and the current or previous treatment protocol has beeneffective, a skilled practitioner may continue the current or previoustreatment protocol to continue regression of the disease, or may chooseto reduce or discontinue the current or previous treatment protocol.Further, the methods of the present invention can also be used todetermine the likelihood of a subject who has undergone a treatmentregimen (e.g., surgery) having a relapse of EAC or BE. Preferably, thesubject would be monitored at a time after the treatment regimen (e.g.,after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3weeks, 1 month, 2 months, 3 months, 4 months 6 months or more than 6months) to determine the likelihood of the subject having a relapse ofEAC or BE.

Typically, therapeutic agents as described for example above will beadministered in pharmaceutical compositions together with apharmaceutically acceptable carrier and in an effective amount toachieve their intended purpose. The dose of active compoundsadministered to a subject should be sufficient to achieve a beneficialresponse in the subject over time such as a reduction in, or relieffrom, the symptoms of EAC or BE. The quantity of the pharmaceuticallyactive compounds(s) to be administered may depend on the subject to betreated inclusive of the age, sex, weight and general health conditionthereof. In this regard, precise amounts of the active compound(s) foradministration will depend on the judgment of the practitioner.

6. Methods of Monitoring Treatment

The present invention can be practiced in the field of predictivemedicine for the purposes of diagnosis or monitoring the presence ordevelopment of a condition selected from EAC or BE in a subject, and/ormonitoring response to therapy efficacy.

The glycospecies profiles of the present invention further enabledetermination of endpoints in pharmacotranslational studies. Forexample, clinical trials can take many months or even years to establishthe pharmacological parameters for a medicament to be used in treatingor preventing EAC or BE. However, these parameters may be associatedwith a glycospecies profile associated with a health state (e.g., HC).Hence, the clinical trial can be expedited by selecting a treatmentregimen (e.g., medicament and pharmaceutical parameters), which resultsin a glycospecies profile associated with the desired health state(e.g., HC). This may be determined for example by (1) providing acorrelation of a reference glycospecies profile with the likelihood ofhaving HC, (2) obtaining a corresponding glycospecies profile of asubject having EAC or BE, after treatment with a treatment regimen,wherein a similarity of the subject's glycospecies profile aftertreatment to the reference glycospecies profile indicates the likelihoodthat the treatment regimen is effective for changing the health statusof the subject to the desired health state (e.g., HC). This aspect ofthe present invention advantageously provides methods of monitoring theefficacy of a particular treatment regimen in a subject (for example, inthe context of a clinical trial) already diagnosed with a conditionselected from EAC or BE. These methods take advantage of glycospeciesbiomarkers that correlate with treatment efficacy, for example, todetermine whether the glycospecies profile of a subject undergoingtreatment partially or completely normalizes during the course of orfollowing therapy or otherwise shows changes associated withresponsiveness to the therapy.

The glycospecies profiles further enable stratification of patientsprior to enrolment in pharmacotranslational studies. For example, aclinical trial can be expedited by selecting a priori patients with aparticular glycospecies profile that would most benefit from aparticular treatment regimen (e.g., medicament and pharmaceuticalparameters). For instance, patient enrolment into a clinical trialtesting the efficacy of a new EAC cancer therapeutic would best includepatients with a glycospecies profile that indicated that they had EACrather than BE, and as such the selected patients would most likelybenefit from the new therapy.

Thus, the invention provides methods of correlating a referenceglycospecies profile with an effective treatment regimen for a conditionselected from EAC or BE, wherein the reference glycospecies profileevaluates at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.)

glycospecies biomarker. These methods generally comprise: (a)determining a sample glycospecies profile from a subject with thecondition prior to treatment (i.e., baseline), wherein the sampleglycospecies profile evaluates for an individual glycospecies biomarkerin the reference glycospecies profile a corresponding glycospeciesbiomarker, and correlating the sample glycospecies profile with atreatment regimen that is effective for treating that condition.

The invention further provides methods of determining whether atreatment regimen is effective for treating a subject with a conditionselected from EAC or BE. These methods generally comprise: (a)correlating a reference glycospecies profile prior to treatment (i.e.,baseline) with an effective treatment regimen for the condition, whereinthe reference glycospecies profile evaluates at least one (e.g., 1, 2,3, 4, 5, 6, 7, 8, 9, 10, etc.) glycospecies biomarker, and (b) obtaininga sample glycospecies profile from the subject after treatment, whereinthe sample glycospecies profile evaluates for an individual glycospeciesbiomarker in the reference glycospecies profile a correspondingglycospecies biomarker, and wherein the sample glycospecies profileafter treatment indicates whether the treatment regimen is effective fortreating the condition in the subject.

The invention can also be practiced to evaluate whether a subject isresponder (i.e., a positive response) or non-responder (i.e., noresponse) to a treatment regimen. This aspect of the invention providesmethods of correlating a glycospecies profile with a positive and/ornegative response to a treatment regimen. These methods generallycomprise: (a) obtaining an glycospecies profile from a subject with acondition selected from EAC or BE following commencement of thetreatment regimen, wherein the glycospecies profile evaluates at leastone (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) glycospecies biomarker,and (b) correlating the glycospecies from the subject with a positiveand/or negative response to the treatment regimen.

The invention also provides methods of determining a positive and/ornegative response to a treatment regimen by a subject with a conditionselected from EAC or BE. These methods generally comprise: (a)correlating a reference glycospecies profile with a positive and/ornegative response to the treatment regimen, wherein the referenceglycospecies profile evaluates at least one (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, etc.) glycospecies biomarker, and (b) determining a sampleglycospecies profile from the subject, wherein the subject's sampleglycospecies profile evaluates for an individual glycospecies biomarkerin the reference glycospecies profile a corresponding glycospeciesbiomarker and indicates whether the subject is responding to thetreatment regimen.

In some embodiments, the methods further comprise determining a firstsample glycospecies profile from the subject prior to commencing thetreatment regimen (i.e., a baseline profile), wherein the first sampleglycospecies profile evaluates at least one (e.g., 1, 2, 3, 4, 5, 6, 7,8, 9, 10, etc.) glycospecies biomarker, and comparing the first sampleglycospecies profile with a second sample glycospecies profile from thesubject after commencement of the treatment regimen, wherein the secondsample glycospecies profile evaluates for an individual glycospeciesbiomarker in the first sample glycospecies profile a correspondingglycospecies biomarker. This aspect of the invention can be practiced toidentify responders or non-responders relatively early in the treatmentprocess, i.e., before clinical manifestations of efficacy. In this way,the treatment regimen can optionally be discontinued, a differenttreatment protocol can be implemented and/or supplemental therapy can beadministered. Thus, in some embodiments, a sample glycospecies profileis obtained within about 2 hours, 4 hours, 6 hours, 12 hours, 1 day, 2days, 3 days, 4 days, 5 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 6weeks, 8 weeks, 10 weeks, 12 weeks, 4 months, six months or longer ofcommencing therapy.

In order that the invention may be readily understood and put intopractical effect, particular preferred embodiments will now be describedby way of the following non-limiting examples.

EXAMPLES Example 1 Identification of Biomarkers for EAC

To identify biomarkers for EAC and BE, the abundance of proteins withaltered glycosylation structures (i.e. different glycospecies) in theserum of healthy patients, patients with BE and patients with EAC wasassessed using lectin-magnetic bead array-coupled mass spectrometry(LeMBA-MS) essentially as described by Choi et al. (Electrophoresis(2011) 32, 3564-3575). A schematic of the biomarker identificationprotocol is shown in FIG. 1.

Materials and Methods Sample Preparation

In the discovery phase, 29 serum samples (TABLE 8), consisting of 10each of BE, EAC and 9 healthy controls (4 confirmed BE-free from Studyof Digestive Health and 6 population controls from Australian CancerStudy), were analysed. One of the control patients subsequentlydeveloped BE, so the data were excluded from further analysis. All ofthe patients were male, reflecting the male-dominance of EAC and BE.

The serum samples were denatured by heating in denaturing buffer (20 mMTris-HCl pH 7.4, 1% w/v SDS, 5% v/v Triton X-100 and 20 mMdithiothreitol (DTT) at 60° C. for 30 minutes, followed by alkylationwith 100 mM iodoacetamide for 1 hr at 37° C., maintaining a darkcondition, prior to dilution for lectin pulldown. 50 μg alkylated serumsample per reaction was incubated with lectin conjugated beads in 100 μlbinding buffer (20 mM Tris-HCl pH 7.4, 300 mM NaCl, 1 mM CaCl₂, 1 mMMnCl₂, 0.05% w/v SDS, 1% v/v Triton X-100) at 4° C. for 1 hour on aplate shaker.

Following the glycoprotein capture, beads were washed three times withbinding buffer, seven times with 50 mM ammonium bicarbonate with threechanges of plates during wash steps. 0.95 μg of sequencing grade trypsinin 20 μl of 50 mM ammonium bicarbonate was added to each reactionmixture and incubated at 37° C. overnight for on-bead trypsin digest. Onthe following day, digested peptides were transferred to a new plate.Beads were washed with equivalent volume of 50 mM ammonium bicarbonateand supernatant was combined with digested peptides. Pooled peptidesamples were dried under the vacuum and plates were stored at −80° C.until further use. Bravo liquid handler (Agilent Technologies) was usedto make the platform high-throughput.

LC-MS/MS and Database search for Biomarker Discovery

The samples are resuspended in 20 μl of 0.1% v/v formic acids forLC-MS/MS. Depending upon lectin used for pull-down, optimal amount oftryptic peptides were subjected to LC-MS using Agilent 6520 QTOF coupledwith a Chip Cube and 1200 HPLC (9 μl were loaded for HAA, HPA and UEA, 6μl for NPL, STL, GNL, 5 μl for BPL, DSA, ECA, MAA, SBA, WFA and WGA, 4μl for AAL, SNA, LPHA, PSA and JAC, 1 μl for EPHA and ConA). The nanopump was set at 0.3 μl/min and the capillary pump at 4 μl/min TheHPLA-chip used contains 160 nl C18 trapping column, and 75μm×150 mm 300Å C18 analytical column (G4240-62010 Agilent Technologies). Buffer A was0.1% v/v formic acid and Buffer B was 90% v/v acetonitrile containing0.1% v/v formic acid. Peptides were eluted from the column usinggradient from 6% B to 46% B at 45 minutes. Nano pump % B was increasedto B at 45.5 min and maintained at the level till 55.5 min. It decreasedto original 6% B at 58.5 minutes. The mass spectrometry was operated in2 GHz extended dynamic range and programmed to acquire 8 precursor MS1spectra per second and 4 MS/MS spectra for each MS spectra. Dynamicexclusion was applied after 2 MS/MS within 0.25 minutes. Exclusion forlectin peptides was applied. The QTOF was tuned and calibrated prior tothe analysis. One hundred femtomold/μl of pre-digested bovine serumalbumin peptides were used as quality control, before and after eachplate. Levels of reference ions 299.2945 and 1221.9906 were maintainedat minimum 5000 and 1000 counts respectively.

To account for experimental variations, 10 pmol chicken ovalbumin, aglycoprotein that binds to every lectin, was spiked in to each sample asinternal standard so as to calculate a normalisation factor for eachidentified protein. LeMBA-MS/MS was then performed by first isolatingserum glycoproteins using a lectin-magnetic bead array that included apanel of 20 lectins (shown in TABLE 9), then performing on-bead trypticdigestion of the glycoproteins followed by LC-MS/MS using an Agilent6520 QTOF couple with a Chip CUBE and 1200 HPLC, as described by Choi etal. (Electrophoresis (2011) 32, 3564-3575). The resulting raw data filewas processed with Spectrum Mill software for database searching againstthe SwissProt human database to identify the glycoproteins.

TABLE 8 DISCOVERY PHASE PATIENT SAMPLES Condition Parameter Healthy (n =9) BE (n = 10) EAC (n = 10) Age in years 66 ± 10 62 ± 15 66 ± 8 (median± SD) Cardiovascular 5 3 3 complications Type 2 diabetes 1 0 1 Gastritis1 1 1 Peptic ulcer 3 2 3 Other 1 2 2 malignancy

TABLE 9 LECTINS USED IN LEMBA-MS Lectin Abbreviation Lectin sourceGeneral reactivity Known target(s) BPL Bauhinia purpurea α/β-D-GalactoseGalβ1-3GalNAc lectin ECA Erythrina cristagalli α/β-D-GalactoseGalβ1-4GlcNAc agglutinin JAC Jacalin α/β-D-Galactose Galα1-6GalNAc andGalβ1-3GalNAc SBA Soybean agglutinin D-N- GalNAcα1-3GalAcetylgalactosamine HPA Helix pomatia D-N- α-GalNAc agglutininAcetylgalactosamine WFA Wisteria floribunda D-N- GalNAcα1-6Gal andagglutinin Acetylgalactosamine GalNAcα1-3GalNAc DSA Datura stramoniumD-N- β1-4GlcNAc lectin Acetylglucosamine oligomers HAA Helix aspersaD-N- α-GlcNAc and α- agglutinin Acetylglucosamine GalNAc STL Solanumtuberosum D-N- GlcNAcβ1-4GlcNAc lectin Acetylglucosamine oligomers WGAWheat germ D-N- GlcNAcβ1-4GlcNAc agglutinin Acetylglucosamine and Neu5AcConA Concanavalin A D-Mannose α-Man, α-Glc, and α- GlcNAc GNL Galanthusnivalis D-Mannose Manα1-3Man lectin NPL Narcissus D-Mannose Manα1-6Manpseudonarcissus lectin AAL Aleuria aurantia α-L-Fucose Fucα1-2, -3, -6linked lectin PSA Pisum sativum α-L-Fucose Fucα1-6GlcNAc of agglutininN-linked glycans UEA Ulex europeus α-L-Fucose Fucα1-2Galβ1- agglutinin-I4GlcNAc MAA Maackia amurensis Sialic acid Neu5Acα2-3Galβ1-3agglutinin-II linkages SNA Sambucus nigra Sialic acid Neu5Acα2-6linkages agglutinin E-PHA Erythroagglutinating Complex specificitiesBisecting GlcNAc phytohemagglutinin L-PHA Leukoagglutinating Complexspecificities Tri/tetra-antennary phytohemagglutinin β1-6GlcNAc

The multi-dimensional data from the LeMBA-MS were stored in theGlycoSelector database as lectin-protein pairs with the measured totalMS1 intensity for the proteins, and the corresponding internal standardfile for the sample. GlycoSelector was then used to perform sampleoutlier detection (FIG. 2) and classification of the glycoproteins bypairwise comparison of the patient groups. Sparse partial least squaresregression-discriminant analysis (sPLS-DA, Le Cao et al. (2011) BMCbioinformatics 12, 253) was used to select a ranked list oflectin-protein pairs that classified between 2 groups. As an example,the sPLS-DA plot in FIG. 3a shows clear separation of BE and EAC usingthe top 100 lectin-protein pairs. Out of the top 100 lectin-proteinpairs, 82 candidates passed the stability cut-off of 0.6 (FIG. 3b ).There was considerable overlap between lectin protein candidatesidentified between healthy vs. BE, BE vs. EAC and healthy vs. EACpatient groups (FIG. 3c ). Each of the 20 lectins used for biomarkerdiscovery showed differential binding with at least one candidate (FIG.3d ). For orthogonal verification of the LeMBA-MS screen byimmunoblotting, we chose two candidates with available antibodies, whichshowed altered binding to AAL lectin. AAL-haptoglobin was one of the topranked stable candidate in sPLS-DA analysis between healthy vs. EAC andBE vs. EAC while AAL-gelsolin was identified using group bindingdifference feature of GlycoSelector as on-off change between BE vs. EACand healthy vs. EAC. Using the same set of discovery serum samples, weperformed AAL lectin pull-down, and measured haptoglobin and gelsolinbinding by immunoblotting. A control serum sample was loaded on everyblot as a normaliser between membranes. Protein level verification byimmunobloting confirmed the MS/MS results (FIG. 3e, 3f ), but showedhigher sensitivity by detecting low levels of gelsolin in all thepatient samples, when some were undetectable by MS/MS.

To feasibly verify a list of candidates identified in biomarkerdiscovery screen in an independent cohort of samples (20 healthy, 21 BE,20 EAC, with all groups having a median age of between 60 and 64 years),multiplexed MRM-MS was optimized for 41 target protein candidates andLeMBA was performed using 6 lectins (AAL, EPHA, JAC, NPL, PSA and WGA).Linearity of the MRM method was determined by spiking range of dilutionsof stable isotope standard (SIS) peptide, spanning 3125 fold dilutionrange into constant amount of LeMBA pull-down sample. The amount of SISpeptide spiked-in for each of four peptides was adjusted in such amanner that response from 1× labeled peptide mix fall within 5-foldrange of the cognate natural peptide. The reproducibility of the dynamicMRM method was determined by running the same sample in triplicate forfour consecutive days. Analysis showed that 86% of the peptides in MRMmethod showed % CV below 10% while 9% of peptides showed % CV between10-20% and only 5% of the peptides were above 20%. Furthermore, % CV forthe entire MRM-MS analysis for SIS as well as natural internal standardchicken ovalbumin peptide was below 20% suggesting robust performance ofthe LeMBA-MRM-MS method. To account for any variation during LeMBApull-down and mass spectrometric measurements, we utilized twonormalization procedures. Firstly, natural ovalbumin peptide intensitywas normalized by spiked-in SIS ovalbumin peptide. Secondly, theintensity of all measured peptides of target proteins was normalizedusing normalized intensity of natural ovalbumin peptide. Univariatestatistical analysis using Kruskal-Wallis tests was performed to assessstatistical significance of each of the candidates. Area under ReceiverOperating Characteristic (AUROC) was calculated to measure thediagnostic potential of each marker and comparison was made betweenhealthy vs. BE, BE vs. EAC and healthy vs. EAC phenotypes (see, TABLE12).

Results

Out of total 246 lectin-protein candidates quantified, 148 candidatesshowed p-value ess than 0.05.

TABLE 10 shows the relative increase or decrease of exemplarydifferentially glycosylated proteins in the serum of patients with EACcompared to healthy patients, BE compared to healthy patients, and inthe serum of patients with EAC compared to patients with BE, i.e. therelative abundance of the specific glycospecies. For example, eightproteins (P00751: complement factor B, P01011: alpha-l-antichymotrypsin,P01031: complement C5,

P02748: complement component C9, P02790: hemopexin, P04003: C4b-bindingprotein alpha chain, P05155: plasma protease Cl inhibitor, P05546:heparin cofactor 2) having glycans that facilitated binding to JAC (i.e.the JAC-binding glycospecies of complement factor B, the JAC-bindingglycospecies of alpha-l-antichymotrypsin, the JAC-binding glycospeciesof complement C5, the JAC-binding glycospecies of complement componentC9, the JAC-binding glycospecies hemopexin, the JAC-binding glycospeciesof C4b-binding protein alpha chain and the JAC-binding glycospecies ofplasma protease Cl inhibitor, and the JAC-binding glycospecies ofheparin cofactor 2) were increased in the serum of patients with EACcompared to healthy patients.

TABLE 10 RELATIVE ABUNDANCE OF GLYCOSPECIES Lectin Proteins (bySwissProt Acc. No.) (glycan) EAC vs HC BE vs HC EAC vs BE AAL ↑ P02748 ↑P00738, P01031, P02748, (Fuc α1,2,3,6 P10643 linked) ↓ P06396 ↓ P04114,P06396 PSA ↑ P00738, P02748 ↑ P00738, P01011, P02748, (Fuc α1- P01031,P10643 6GlcNAc) ↓ P06396 ↓ P06396 EPHA ↑ P00738, P02748 ↑ P01023 ↑P00738, P01011, P02748, (Bisecting P02787, P10643 GlcNAc) ↓ P01023,P02765, ↓ P06396 P06396 JAC ↑ P00751, P01011, ↑ P04114 ↑ P00738, P00751,P01009, (Galβ1- P01031, P02748, P02790, P01011, P01031, P02748, 6GalNAc,P04003, P05155, P05546 P04217, P10643 Galβ1- ↓ P06396 ↓ P06396 3GalNac)NPL ↑ P00738, P01011, ↑ P04114 ↑ P02748 (Mannose P02748, P04003 α1-3Man)↓ P04114, P06396, P43652 WGA ↑ P00738, P02748, ↑ P00738, P02748(GlcNAcβ1- P01011 4GlcNAc and ↓ P06396 Neu5Ac)

FIG. 4 shows the relative abundance of exemplary glycospecies that weredemonstrated to be significantly increased or decreased in at least twoof EAC, BE and healthy subjects, and which could thus be used todistinguish between healthy, pre-cancer (BE) patients and EAC patients,and therefore determine the likelihood of the presence or absence ofEAC. For example, the AAL-binding gelsolin (P06396) and PSA-bindinggelsolin glycospecies were present in the serum of EAC patients atsignificantly reduced levels compared to the serum of BE patients.Conversely, the AAL-binding complement component C9 (P02748),AAL-binding complement component C9, and EPHA-binding haptoglobin(P00738) glycospecies were present in the serum of EAC patients atsignificantly increased levels compared to the serum of BE patients.FIGS. 3 show the relative abundance of exemplary glycospecies that weredemonstrated to have a statistically significant increase or decrease inamount (as assessed using an ANOVA-Tukey test) in the serum of BEpatients compared to healthy patients, and which could thus be used todistinguish healthy patients from pre-cancer patients with EAC.

FIG. 4 shows LeMBA-MS data for gelsolin. Specifically, the Y-axis showsrelative abundance (note the log scale), and the X-axis shows thebinding to each of the 20 lectins, grouped into general reactivitygroups. Boxed lectins show statistically significantly different bindingbetween BE and EAC groups (*p<0.05, Student's t-test). The graph clearlyshows that the gelsolin glycospecies that most clearly distinguishbetween BE and EAC based on their relative abundance, and in particularNPL-binding gelsolin, JAC-binding gelsolin, PSA-binding gelsolin andGNL-binding gelsolin, which are significantly reduced in the serum ofEAC patients compared to BE patients, suggesting a reduction in multipleglycan structures on gelsolin in EAC patients.

Example 2 Detecting Combinations of Glycospecies

In order to determine whether a particularly robust set of markers couldselected in order to improve the determination of the likelihood of thepresence or absence of BE or EAC in a subject, combinations ofglycospecies identified from the relevant table (TABLES 1, 4, and 6)were analysed.

It was found that the power of the diagnostic test could be enhanced bymeasuring a panel of two, three, four, five, or more than five markers(see, TABLE 11). Notably, when using four glycospecies (JAC-bindingcomplement component C9, EPHA-binding alpha-1B-glycoprotein,EPHA-binding gelsolin, WGA-binding angiotensin andalpha-2-macroglobulin) are measured between subjects with EAC andhealthy controls, an AUC of up to 98.25% can be achieved.

TABLE 11 AUC Glycospecies (EAC v Healthy) P02748_JAC 0.775 P02748_JACP04217_EPHA 0.86 P02748_JAC P04217_EPHA P06396_EPHA 0.93 P02748_JACP04217_EPHA P06396_EPHA P01019_WGA 0.9825 P02748_JAC P04217_EPHAP06396_EPHA P01019_WGA P01023_NPL 0.98 P02748_JAC 0.775 P02748_JACP06396_EPHA 0.8475 P02748_JAC P06396_EPHA P02748_WGA 0.8525 P02748_JACP06396_EPHA P02748_WGA P02748_NPL 0.8525 P02748_JAC P06396_EPHAP02748_WGA P02748_NPL P06396_SNA 0.86 Glycospecies (EAC v BE) P02748_AAL0.8525 P02748_AAL P02748_JAC 0.835 P02748_AAL P02748_JAC P02748_PSA0.8375 P02748_AAL P02748_JAC P02748_PSA P02748_EPHA 0.8425 P02748_AALP02748_JAC P02748_PSA P02748_EPHA P02748_WGA 0.8375 P02748_AAL 0.8525P02748_AAL P04114_NPL 0.91 P02748_AAL P04114_NPL P04217_EPHA 0.9625P02748_AAL P04114_NPL P04217_EPHA P01781_PSA 0.975 P02748_AAL P04114_NPLP04217_EPHA P01781_PSA P0C0L5_WGA 0.985

TABLE 12 DIFFERENTIAL EXPRESSION OF GLYCOSPECIES Glycospecies Kruskal-(Lectin-SwissProt Wallis No) p-value AUROC BE vs EAC AAL_P00738 0.03980.69 EPH_P00738 0.0200 0.715 JAC_P00738 0.0483 0.6825 PSA_P00738 0.04830.6825 WGA_P00738 0.0483 0.6825 JAC_P00751 0.0398 0.69 JAC_P01009 0.04530.685 EPH_P01011 0.0265 0.705 JAC_P01011 0.0102 0.705 PSA_P01011 0.04250.6875 AAL_P01031 0.0483 0.6825 JAC_P01031 0.0398 0.69 PSA_P01031 0.04530.685 AAL_P02748 0.0001 0.8525 EPH_P02748 0.0003 0.8375 JAC_P027480.0007 0.8125 NPL_P02748 0.0049 0.76 PSA_P02748 0.0008 0.81 WGA_P027480.0032 0.7725 EPH_P02787 0.0326 0.6975 AAL_P04114 0.0483 0.6825NPL_P04114 0.0248 0.7075 JAC_P04217 0.0483 0.6825 AAL_P06396 0.00870.7425 EPH_P06396 0.0186 0.7175 JAC_P06396 0.0305 0.7 NPL_P06396 0.01730.72 PSA_P06396 0.0483 0.6825 WGA_P06396 0.0128 0.73 AAL_P10643 0.00630.7525 EPH_P10643 0.0398 0.69 JAC_P10643 0.0094 0.74 PSA_P10643 0.00190.7875 NPL_P43652 0.0483 0.6825 EAC vs HC AAL_P00738 0.0583 0.675EPH_P00738 0.0305 0.7 NPL_P00738 0.0349 0.695 PSA_P00738 0.0425 0.6875WGA_P00738 0.0215 0.7125 JAC_P00751 0.0373 0.6925 JAC_P01011 0.0305 0.7NPL_P01011 0.0305 0.7 WGA_P01011 0.0080 0.745 EPH_P01023 0.0186 0.7175JAC_P01031 0.0483 0.6825 AAL_P02748 0.0161 0.7225 EPH_P02748 0.02650.705 JAC_P02748 0.0029 0.775 NPL_P02748 0.0074 0.7475 PSA_P02748 0.01610.7225 WGA_P02748 0.0049 0.76 EPH_P02765 0.0483 0.6825 JAC_P02790 0.02000.715 JAC_P04003 0.0138 0.7275 JAC_P05155 0.0200 0.715 JAC_P05546 0.04830.6825 AAL_P06396 0.0265 0.705 EPH_P06396 0.0014 0.795 JAC_P06396 0.02000.715 PSA_P06396 0.0110 0.735 BE vc HC EPH_P01023 0.0248 0.7075JAC_P04114 0.0305 0.7 NPL_P04114 0.0215 0.7125

While some gelsolin glycospecies were present at similar levels in theserum of healthy, BE and EAC patients (e.g. WGA-binding gelsolin),others were present at statistically different levels in the variousgroups. For example, glycospecies characterised as being in the serum ofEAC patients at significantly reduced amounts compared to BE patients(statistical analysis using the Student's t-test), but were not presentin significantly different amounts between healthy and BE patients.These results indicated that there was a reduction in many differentglycan structures, including various glycans containing D-Mannose,D-N-Acetylglucosamine, α/β-D-Galactose and α-L-Fucose, in plasmagelsolin proteins of EAC patients compared to BE patients.

To exclude the possibility that loss of glycosylated gelsolin in EAC wasdue to an overall loss of gelsolin protein in the serum, immunoblottingwith an anti-gelsolin antibody was performed to measure the level tototal gelsolin in some of the serum samples. As shown in FIG. 5, therewas a non-significant trend towards an increased level of gelsolin inthe serum of BE patients compared to healthy and EAC patients, while theamount of AAL-binding and PSA-binding gelsolin was significantly reducedin EAC patients compared to healthy patients (as indicated by gelsolinbinding to the two fucose-reactive lectins, AAL and PSA).

Example 3 Validation of Glycospecies Biomarkers

In order to demonstrate that glycospecies biomarkers can be reliablydetermine the likelihood of a subject having a relevant condition, anumber of the biomarkers were selected for validation in a separate anddistinct cohort of subjects.

TABLE 13 shows the relative increase or decrease of exemplarydifferentially glycosylated proteins in the serum of patients with EACcompared to healthy patients, BE compared to healthy patients, and inthe serum of patients with EAC compared to patients with BE, i.e. therelative abundance of the specific glycospecies. For example, sixproteins (P00738: haptoglobin, P00751: complement factor B, P01011:alpha-1-antichymotrypsin, P02748: complement component C9, P09871:complement Cls subcomponent, and P10643: complement component C7) havingglycans that facilitated binding to EPHA (i.e. the EPHA-bindingglycospecies of haptoglobin, the EPHA-binding glycospecies of complementfactor B, the EPHA-binding glycospecies of alpha- 1-antichymotrypsin,the JAC-binding glycospecies of complement component C9, theEPHA-binding glycospecies complement Cls subcomponent, and theEPHA-binding glycospecies of complement component C7) were increased inthe serum of patients with EAC compared to healthy patients

TABLE 13 Lectin Proteins (by SwissProt Acc. No.) (glycan) EAC vs HC BEvs HC EAC vs BE AAL ↑ P01009; P01011; ↑ P51884 ↑ P02748, P00734 (Fucα1,2,3,6 P02748; P04217; linked) P09871; P10643; P19652 ↓ P02679;P02753; ↓ P02753 P06396; P07225; P35858; P43652; Q96PD5 EPHA ↑ P00738;P00751; ↑ P51884 ↑ P00751, P02748 (Bisecting P01011; P02748; GlcNAc)P09871; P10643 ↓ P02679; P02765; ↓ P00734; P02751; ↓ P02749, P06396,P51884, P06396; P33151; Q15166 P68871; Q96PD5 JAC ↑ P00738; P01009; ↑P00450, P00734; P01011,, (Galα1- P01011, P02748, P02748, P02790, P04217,6GalNAc, P05155, P09871 P05155, P05156, P08603, P09871, P20851, Q14624Galβ1- ↓ P02647; P02675; ↓ O95445; P00734; 3GalNac) P02679; P02753;P01019; P02647; P02675; P02765; P02787; P03952; P04114; P04196; P06396;P07225; P07225; P07357; P08603; P03952; P06396; P08697; P29622; P43652;P07225; P29622; P68871 P33151; P35858; P68871; Q7Z7A1; Q96PD5 NPL ↑P00738, P01009; ↑ P02749 ↑ P02748 (Mannose α1- P01011, P02748, 3Man)P02790; P04217 ↓ P02647; P02765; ↓ P04114; P08697; ↓ P06396 P02787,P06396; P33151; P68871 P07225; P27169; P33151; P35858; P43652; P68871;Q15166; Q96PD5

Materials and Methods Study Design and Sample Information

Serum samples were collected from consenting patients undergoing uppergastrointestinal at Ochsner Health Systems, New Orleans, USA. The studywas approved by the Human Research Ethics Committees of Ochsner HealthSystems and the University of Queensland. Patient diagnosis wasaccording to current practice, endoscopy with histology of biopsysamples, and classified as BE (Barrett's esophagus), EAC (esophagealadenocarcinoma) or healthy (i.e., non-BE/EAC). TABLEe 14 describes theclinical characteristics of patients. Samples were randomized prior toall experiments. The samples were stored at −80° C. until use.

TABLE 14 BE EAC HC Variables Sample size 12  10  16  Gender (% Male) 83%80% 75% Age (Median ± SD) 71 ± 10 63 ± 10 66 ± 11 Body mass indexHealthy (<25) 7 2 2 Overweight (25-30) 2 5 4 Obese (>=30) 3 3 10 

Materials and Methods

MyOne™ Tosyl activated Dynabeads were from Life Technologies. LectinsAAL, EPHA, JAC, and NPL were from Vector Laboratories. Modifiedsequencing grade trypsin was from Promega. Triton X-100 and sodiumdodecyl sulfate solution were from Bio-rad. Tris base, glycine, andsodium chloride were from Amresco. Disodium hydrogen phosphatedihydrate, sodium dihydrogen phosphate dihydrate, and calcium chloridedihydrate were from Ajax Finechem. Manganese chloride was from Univar.Acetonitrile CHROMASOLV® gradient grade was from Sigma. All otherreagents including lectins not listed above were from Sigma unlessotherwise specified.

Methodologies

Serum samples were screened using LeMBA-MRM-MS assay with four lectins(AAL, EPHA, JAC, and NPL) as reported previously in Shah et al. 2015Mol. Cell. Proteomics 14, 3023-3039.

Lectin Magnetic Bead Array (LeMBA)

Lectins were conjugated with magnetic beads as described previously inShah et al. Mol. Cell. Proteomics 14, 3023-3039. (2, 3). For each pulldown experiment, lectin-beads (AAL, EPHA, JAC, and NPL) were arrayed ineach well of a 96 well plate. Serum samples (allowing 50 μg perpull-down as measured by BCA protein assay) were spiked with 10 pmolovalbumin per reaction as an internal standard. The serum proteinmixture was denatured and reduced using denaturing buffer (20 mMTris-HC1 pH 7.4, 1% w/v SDS, 5% v/v Triton X-100 and 20 mMDithiothreitol) at 60° C. for 30 min followed by alkylation with 100 mMiodoacetamide for 1 hr at 37° C. in the dark. Alkylated serum sample (50μg per reaction) was incubated with lectin conjugated beads in 100 μlbinding buffer (20 mM Tris-HCl pH 7.4, 300 mM NaCl, 1 mM CaCl₂, 1 mMMnCl₂, 0.05% w/v SDS, 1% v/v Triton X-100) at 4° C. for 1 hr on a plateshaker to allow glycoprotein-lectin binding. Beads were then washedsequentially with (i) binding buffer 3 times and (ii) 50 mM ammoniumbicarbonate seven times, including three plate changes in-betweenwashes. For on-bead trypsin digest, 0.95 μg of sequencing grade trypsinin 20 μl of 50 mM ammonium bicarbonate was added to each reactionmixture and incubated at 37° C. overnight. The next day, digestedpeptides were transferred to a new plate. Beads were washed with anequivalent volume of 50 mM ammonium bicarbonate, and the supernatant wascombined with digested peptides. Peptide samples were vacuum-dried andthe plates were stored at −80° C. until further use. Bravo liquidhandler (Agilent Technologies) was used to make the platform highthroughput.

MRM-MS

Multiple reaction monitoring-mass spectrometry (MRM-MS) assay wasperformed on Agilent Technologies 6490 triple quadrupole massspectrometer coupled with 1290 standard-flow infinity UHPLC fitted witha standard-flow ESI (Jet Stream) source. A customized MRM-MS assay for114 target proteins was developed and used to measure four lectinpull-downs (AAL, EPHA, JAC, and NPL) for each patient sampleindependently. Detail strategy for MRM-MS assay development wasdescribed in Shah et al. 2015 Mol. Cell. Proteomics 14, 3023-3039.

LC Method Development

The UHPLC system consisted of a reverse phase chromatographic column

AdvanceBio Peptide Mapping (150×2.1 mm i.d., 2.7 μm, part number653750-902, Agilent Technologies) with a 5 mm long guard column. Mobilephase A consisted of 0.1% formic acid, and mobile phase B consisted of100% acetonitrile and 0.1% formic acid. The UHPLC system was operated at60° C., with a flow rate of 0.4 mL/min The gradient used for peptideseparation was as follows: 3% B at 0 min; 35% B at 40 min; 95% B at40.50 min; 95% B at 44.50 min; 3% B at 45 min; followed by conditioningof columns for 4 min at 3% B before injecting the next sample.

Mass Spectrometer Settings

Agilent 6490 triple quadrupole mass spectrometer was operated inpositive ion mode and controlled by Agilent's MassHunter Workstationsoftware (version B.06.00 build 6.0.6025.4 SP4). The MRM acquisitionparameters were 150 V high pressure RF, 60 V low pressure RF, 4000 Vcapillary voltage, 300 V nozzle voltage, 11 L/min sheath gas flow at atemperature of 250° C., 15 L/min drying gas flow at a temperature of250° C., 30 psi nebulizer gas flow, unit resolution (0.7 Da full widthat half maximum in the first quadrupole (Q1) and the third quadrupole(Q3), and 200 V delta EMV (+).

Screening Samples for LeMBA-MRM-MS Qualification

Lectin-beads sufficient for biomarker qualification experiments weremade in a single batch to minimize experimental variation. Serum sampleswere randomized for LeMBA-MRM-MS experiments. Peptide samples werespiked with SIS peptide mixture containing 50 femtomole of SPAFTDLHLR,AVEVLPK, and LTPLYELVK each, 100 femtomole of LSPIYNLVPVK, 200 femtomoleof NLAVSQVVHK, 500 femtomole of VASMASEK,

ISQAVHAAHAEINEAGR, and GSFEFPVGDAVSK each, and 1000 femtomole ofVTSIQDWVQK, and LPPNVVEESAR each.

Data Processing and Statistical Analysis

Raw data from MRM-MS experiment was processed using Skyline. All peakswere manually checked for correct integration, and peak area for eachpeptide (sum of all transitions) was exported for further analysis.Firstly, raw peptide intensity were normalized according to SIS peptidemixture. Followed by the two step normalization described in Shah et al.2015 Mol. Cell. Proteomics 14, 3023-3039. Univariate, multivariate andROC curve analyses were performed using Shiny mixOmics (available on theworld wide web at mixomics-projects.di.uq.edu.au/Shiny-dev/) asdescribed in Shah et al. 2015 Mol. Cell. Proteomics 14, 3023-3039.

Example 4 Detecting Additional Combinations of Glycospecies Biomarkers

In order to determine whether an additional robust set of markers couldselected in order to assist with or improve the determination of thelikelihood of the presence or absence of BE or EAC in a subject,combinations of glycospecies identified from the relevant table (TABLES3, 5 and 7) were analysed.

It was found that the power of the diagnostic test could be enhanced bymeasuring a panel of two or more markers. Notably, when sevenglycospecies are measured between subjects with EAC and subjects withBE, an AUC of up to 95% can be achieved (see, TABLE 15; and FIG. 6).

TABLE 15 Comparison Glycospecies Biomarkers AUROC BE vs EAC (1)P00734_AAL; (2) P02748_JAC; 0.9500 (3) P02748_EPH (4) P06396_NPL; (5)P00734_JAC; (6) P00450_JAC; (7) P02748_NPL HC vs EAC (1) P01011_JAC; (2)P02748_EPH; 1.000 (3) P01011_NPL; (4) P02748_JAC; (5) P01011_EPH; (6)P01011_AAL; (7) P02748_NPL; (8) P09871_EPH; (9) P02748_AAL; (10)P10643_EPH; (11) Q96PD5_NPL HC vs BE (1) P51884-AAL; (2) P51884-EPH0.9111

The disclosure of every patent, patent application, and publicationcited herein is hereby incorporated herein by reference in its entirety.

The citation of any reference herein should not be construed as anadmission that such reference is available as “Prior Art” to the instantapplication.

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. Those of skill in the artwill therefore appreciate that, in light of the instant disclosure,various modifications and changes can be made in the particularembodiments exemplified without departing from the scope of the presentinvention. All such modifications and changes are intended to beincluded within the scope of the appended claims.

1.-54. (canceled)
 55. A composition comprising a biological sample froma subject having or suspected of having Barrett's esophagus (BE) oresophageal adenocarcinoma (EAC) comprising a glycospecies of at leastone glycoprotein; and one or more reagents for determining the level ofsaid glycospecies, wherein the at least one glycoprotein comprisescomplement C9.
 56. The composition of claim 55, wherein the compositioncomprises a glycospecies of at least one other glycoprotein selectedfrom the group consisting of gelsolin, N-acetylmuramoyl-L-alanineamidase, afamin, alpha-1-antichymotrypsin, alpha-1-antitrypsin,alpha-1B-glycoprotein, alpha-2-HS -glycoprotein, alpha-2-macroglobulin,apolipoprotein B-100, beta-2-glycoprotein 1, ceruloplasmin, complementC1q subcomponent subunit B, complement C4b-binding protein alpha chain,complement C5, complement C7, complement factor B, haptoglobin,hemopexin, kallistatin, plasma kallikrein, plasma protease C 1inhibitor, serum paraoxonase/arylesterase 1, serum paraoxonase/lactonase3, retinol-binding protein 4, complement C4b-binding protein beta chainand serotransferrin; and a reagent for determining the level of saidglycospecies.
 57. The composition of claim 56, wherein the at least oneother glycoprotein comprises gelsolin.
 58. The composition of claim 55,wherein the one or more reagents comprise an antibody.
 59. Thecomposition of claim 55, wherein the one or more reagents comprise aglycan-binding molecule.
 60. The composition of claim 59, wherein theglycan-binding molecule is selected from the group consisting of alectin, a glycospecific antibody, a glycospecific aptamer, aglycospecific peptide and a glycospecific small molecule.
 61. Thecomposition of claim 59, wherein the glycan-binding molecule is a lectinselected from the group consisting of Aleuria aurantia lectin (AAL),Pisum sativum agglutinin (PSA), erythroagglutinating phytohemagglutinin(EPHA), jacalin (JAC), Narcissus pseudonarcissus lectin (NPL) and wheatgerm agglutinin (WGA).
 62. The composition of claim 55, whereinindividual glycospecies defined by a glycan-binding molecule and theglycoprotein to which it binds are selected from the group consisting ofAAL:complement C9, AAL:gelsolin, AAL:N-acetylmuramoyl-L-alanine amidase,AAL:retinol-binding protein 4, PSA:haptoglobulin, PSA:complement C9,PSA:gelsolin, EPHA:haptoglobin, EPHA:complement factor B,EPHA:complement C9, EPHA:gelsolin, EPHA:alpha-2-macroglobulin,EPHA:alpha-2-HS -glycoprotein, EPHA:N-acetylmuramoyl-L-alanine amidase,EPHA: serum paraoxonase/lactonase 3, JAC:alpha-l-antitrypsin,JAC:ceruloplasmin, JAC:complement factor B,JAC:alpha-1-antichymotrypsin, JAC:complement C5, JAC:complement C9,JAC:hemopexin, JAC:C4b-binding protein alpha chain,JAC:N-acetylmuramoyl-L-alanine amidase, JAC:plasma kallikrein,JAC:plasma protease C 1 inhibitor, JAC:heparin cofactor 2, JAC:gelsolin,NPL:ceruloplasmin, NPL:gelsolin, NPL:haptoglobin,NPL:alpha-1-antichymotrypsin, NPL:complement C9, NPL:C4b-binding proteinalpha chain, NPL:N-acetylmuramoyl-L-alanine amidase, NPL: serumparaoxonase/arylesterase 1, NPL: serum paraoxonase/lactonase 3,WGA:haptoglobin, WGA:alpha-1-antichymotrypsin, WGA:complement C1qsubcomponent subunit B and WGA:complement C9.
 63. The composition ofclaim 55, wherein individual glycospecies defined by a glycan-bindingmolecule and the glycoprotein to which it binds are selected from thegroup consisting of AAL:haptoglobin, AAL:complement C5, AAL:complementC9, AAL:complement C7, AAL:apolipoprotein B-100, AAL:gelsolin,AAL:retinol-binding protein 4, PSA:haptoglobin,PSA:alpha-l-antichymotrypsin, PSA:complement 5, PSA:complement C9,PSA:complement C7, PSA:gelsolin, EPHA:haptoglobin,EPHA:alpha-1-antichymotrypsin, EPHA:complement C9, EPHA:serotransferrin,EPHA:complement factor B, EPHA:complement C7, EPHA:gelsolin, EPHA:serumparaoxonase/lactonase 3, JAC:haptoglobin, JAC:complement factor B,JAC:alpha-1-antitrypsin, JAC: alpha-1-antichymotrypsin,JAC:ceruploplasmin, JAC:complement C5, JAC:complement C9,JAC:alpha-1B-glycoprotein, JAC:complement C7, JAC:gelsolin,NPL:complement C9, NPL: apolipoprotein B-100, NPL:gelsolin, NPL:afamin,WGA:haptoglobin, WGA:complement C9 and WGA:gelsolin.
 64. The compositionof claim 56, wherein the at least one other glycoprotein is selectedfrom the group consisting of gelsolin, alpha-1-antitrypsin, serumparaoxonase/arylesterase 1, serum paraoxonase/lactonase 3 and plasmakallikrein.
 65. The composition of claim 64, wherein individualglycospecies defined by a glycan-binding molecule and the glycoproteinto which it binds, are selected from the group consisting ofJAC:complement C9, NPL:complement C9, EPHA:complement C9, EPHA:gelsolin,JAC:gelsolin, NPL:gelsolin, NPL:serum paraoxonase/arylesterase 1, NPL:serum paraoxonase/lactonase 3, EPHA:serum paraoxonase/lactonase 3,JAC:alpha-1-antitrypsin and JAC:plasma kallikrein.
 66. The compositionof claim 57, wherein individual glycospecies defined by a glycan-bindingmolecule and the glycoprotein to which it binds are selected from thegroup consisting of AAL:complement C9, AAL:gelsolin, PSA:complement C9,PSA:gelsolin, EPHA:complement C9, EPHA:gelsolin, JAC:complement C9,JAC:gelsolin, NPL:complement C9 and WGA:complement C9.
 67. Thecomposition of claim 57, wherein individual glycospecies defined by aglycan-binding molecule and the glycoprotein to which it binds areselected from the group consisting of AAL:complement C9, AAL:gelsolin,PSA:complement C9, PSA:gelsolin, EPHA:complement C9, EPHA:gelsolin,JAC:complement C9, JAC:gelsolin, NPL:complement C9, NPL:gelsolin,WGA:complement C9 and WGA:gelsolin.
 68. The composition of claim 55,wherein the biological sample is a serum, plasma or blood sample.
 69. Amethod of treating esophageal adenocarcinoma (EAC) in a subject,comprising: a) determining in a sample from the subject the level of aglycospecies of at least one glycoprotein, which glycospecies isdifferentially expressed between EAC and one or more other conditionsselected from healthy condition (HC) and Barrett's esophagus (BE), anddetermining a likelihood of the subject having EAC based on whether therespective levels of the individual glycospecies are above or below acorresponding predetermined threshold that correlates with the presenceof EAC, wherein the at least one glycoprotein comprises complement C9;and b) exposing the subject to a treatment regimen for EAC.
 70. Themethod of claim 69, wherein the method further comprises determining thelevel of a glycospecies of at least one other glycoprotein selected fromthe group consisting of gelsolin, N-acetylmuramoyl-L-alanine amidase,afamin, alpha-1-antichymotrypsin, alpha-1-antitrypsin,alpha-1B-glycoprotein, alpha-2-HS -glycoprotein, alpha-2-macroglobulin,apolipoprotein B-100, beta-2-glycoprotein 1, ceruloplasmin, complementC1q subcomponent subunit B, complement C4b-binding protein alpha chain,complement C5, complement C7, complement factor B, haptoglobin,hemopexin, kallistatin, plasma kallikrein, plasma protease C1 inhibitor,serum paraoxonase/arylesterase 1, serum paraoxonase/lactonase 3,retinol-binding protein 4, complement C4b-binding protein beta chain andserotransferrin.
 71. The method of claim 70, wherein the at least oneother glycoprotein comprises gelsolin.
 72. The method of claim 69,wherein the level of an individual glycospecies is determined bycontacting the sample with a glycan-binding molecule under conditionsthat permit binding of the glycan-binding molecule to the glycospecies.73. The method of claim 72, wherein the glycan-binding molecule isselected from the group consisting of a lectin, a glycospecificantibody, a glycospecific aptamer, a glycospecific peptide and aglycospecific small molecule.
 74. The method of claim 73, wherein theglycan-binding molecule is a lectin selected from the group consistingof Aleuria aurantia lectin (AAL), Pisum sativum agglutinin (PSA),erythroagglutinating phytohemagglutinin (EPHA), jacalin (JAC), Narcissuspseudonarcissus lectin (NPL) and wheat germ agglutinin (WGA).
 75. Themethod of claim 74, wherein individual glycospecies defined by aglycan-binding molecule and the glycoprotein to which it binds and whichare differentially expressed between EAC and HC are selected from thegroup consisting of AAL:complement C9, AAL:gelsolin,AAL:N-acetylmuramoyl-L-alanine amidase, AAL:retinol-binding protein 4,PSA:haptoglobulin, PSA:complement C9, PSA:gelsolin, EPHA:haptoglobin,EPHA:complement factor B, EPHA:complement C9, EPHA:gelsolin,EPHA:alpha-2-macroglobulin, EPHA:alpha-2-HS-glycoprotein,EPHA:N-acetylmuramoyl-L-alanine amidase, EPHA: serumparaoxonase/lactonase 3, JAC:alpha-1-antitrypsin, JAC: ceruloplasmin,JAC:complement factor B, JAC:alpha-1-antichymotrypsin, JAC:complementC5, JAC:complement C9, JAC:hemopexin, JAC:C4b-binding protein alphachain, JAC:N-acetylmuramoyl-L-alanine amidase, JAC:plasma kallikrein,JAC:plasma protease C₁ inhibitor, JAC:heparin cofactor 2, JAC:gelsolin,NPL:ceruloplasmin, NPL:gelsolin, NPL:haptoglobin, NPL:alpha-1-antichymotrypsin, NPL:complement C9, NPL:C4b-binding proteinalpha chain, NPL:N-acetylmuramoyl-L-alanine amidase, NPL: serumparaoxonase/arylesterase 1, NPL: serum paraoxonase/lactonase 3,WGA:haptoglobin, WGA:alpha-1-antichymotrypsin, WGA:complement C1qsubcomponent subunit B and WGA:complement C9.
 76. The method of claim74, wherein individual glycospecies defined by a glycan-binding moleculeand the glycoprotein to which it binds and which are differentiallyexpressed between EAC and BE are selected from the group consisting ofAAL:haptoglobin, AAL:complement C5, AAL:complement C9, AAL:complementC7, AAL:apolipoprotein B-100, AAL:gelsolin, AAL:retinol-binding protein4, PSA:haptoglobin, PSA:alpha-1-antichymotrypsin, PSA:complement 5,PSA:complement C9, PSA:complement C7, PSA:gelsolin, EPHA:haptoglobin,EPHA: alpha-1-antichymotrypsin, EPHA:complement C9,EPHA:serotransferrin, EPHA:complement factor B, EPHA:complement C7,EPHA:gelsolin, EPHA:serum paraoxonase/lactonase 3, JAC:haptoglobin,JAC:complement factor B, JAC:alpha-1-antitrypsin, JAC:alpha-1-antichymotrypsin, JAC:ceruploplasmin, JAC:complement C5,JAC:complement C9, JAC:alpha-1B-glycoprotein, JAC:complement C7,JAC:gelsolin, NPL:complement C9, NPL: apolipoprotein B-100,NPL:gelsolin, NPL:afamin, WGA:haptoglobin, WGA:complement C9 andWGA:gelsolin.
 77. The method of claim 70, wherein the at least one otherglycoprotein is selected from the group consisting of gelsolin,alpha-1-antitrypsin, serum paraoxonase/arylesterase 1, serumparaoxonase/lactonase 3 and plasma kallikrein.
 78. The method of claim77, wherein individual glycospecies defined by a glycan-binding moleculeand the glycoprotein to which it binds, are selected from the groupconsisting of JAC:complement C9, NPL:complement C9, EPHA:complement C9,EPHA:gelsolin, JAC:gelsolin, NPL:gelsolin, NPL:serumparaoxonase/arylesterase 1, NPL: serum paraoxonase/lactonase 3,EPHA:serum paraoxonase/lactonase 3, JAC:alpha-1-antitrypsin andJAC:plasma kallikrein.
 79. The method of claim 71, wherein individualglycospecies defined by a glycan-binding molecule and the glycoproteinto which it binds and which are differentially expressed between EAC andHC are selected from the group consisting of AAL:complement C9,AAL:gelsolin, PSA:complement C9, PSA:gelsolin, EPHA:complement C9,EPHA:gelsolin, JAC:complement C9, JAC:gelsolin, NPL:complement C9 andWGA:complement C9.
 80. The method of claim 71, wherein individualglycospecies defined by a glycan-binding molecule and the glycoproteinto which it binds and which are differentially expressed between EAC andBE are selected from the group consisting of AAL:complement C9,AAL:gelsolin, PSA:complement C9, PSA:gelsolin, EPHA:complement C9,EPHA:gelsolin, JAC:complement C9, JAC:gelsolin, NPL:complement C9,NPL:gelsolin, WGA:complement C9 and WGA:gelsolin.
 81. The method ofclaim 69, wherein the treatment regimen comprises surgery, radiotherapyor chemotherapy.
 82. The method of claim 81, wherein the treatmentregimen comprises surgery, and said surgery removes all or part of theesophagus.